Taenia solium

FACULTY OF HEALTH AND MEDICAL SCIENCES UNIVERSITY OF COPENHAGEN

FACULTY OF HEALTH AND MEDICAL SCIENCES UNIVERSITY OF COPENHAGEN

PhD Thesis Uffe Christian Braae  

PhD Thesis

 

Epidemiology and control of Taenia solium in Africa

Uffe Christian Braaeand control of Taenia solium in Africa Epidemiology  

 

Academic advisors: Maria Vang Johansen and Pascal Magnussen Submitted: 30 th November 2016

Academic advisors: Maria Vang Johansen and Pascal Magnussen Submitted: 30 th November 2016

Epidemiology and control of Taenia solium in Africa PhD Thesis 2017 © Uffe Christian Braae ISBN: 978-87-93476-83-7 Printed by SL grafik, Frederiksberg C, Denmark (slgrafik.dk)

PhD Thesis

Uffe Christian Braae

Enrolled under the school of Public Health & Epidemiology at: Section for Parasitology and Aquatic Diseases Department of Veterinary and Animal Sciences Faculty of Health and Medical Sciences Dyrlægevej 100, 1870 Frederiksberg C, Denmark

This thesis has been submitted to the Graduate School of The Faculty of Health and Medical Sciences, University of Copenhagen [30-11-2016]

2016

Supervisors

Assessment committee

Professor Maria Vang Johansen

Professor Stig Milan Thamsborg (Chairman)

(Supervisor)

Section for Parasitology and Aquatic Diseases

Section for Parasitology and Aquatic Diseases

Department of Veterinary and Animal

Department of Veterinary and Animal

Sciences

Sciences

Faculty of Health and Medical Sciences

Faculty of Health and Medical Sciences

Dyrlægevej 100, 1870-DK Frederiksberg C,

Dyrlægevej 100, 1870-DK Frederiksberg C,

Denmark

Denmark Associate Professor Pascal Magnussen (Co-

Professor Tine Hald

supervisor)

Research Group for Genomic Epidemiology

Section for Parasitology and Aquatic Diseases

National Food Institute

Department of Veterinary and Animal

Technical University of Denmark

Sciences

Søltofts Plads, 2800 Kgs. Lyngby, Denmark

Faculty of Health and Medical Sciences Dyrlægevej 100, 1870-DK Frederiksberg C,

Professor Eric Fèvre

Denmark

Institute of Infection and Global Health,

and

University of Liverpool

Centre for Medical Parasitology

Leahurst Campus, Chester High Road,

Faculty of Health and Medical Sciences

Neston, CH64 7TE, United Kingdom

Bartholinsgade 2, 1356-DK Copenhagen K,

and

Denmark

International Livestock Research Institute Old Naivasha Road, PO Box 30709-00100, Nairobi, Kenya

Table of Contents PREFACE ........................................................................................................................................... I ACKNOWLEDGEMENTS............................................................................................................ IV SUMMARY .................................................................................................................................... VII SAMMENDRAG (DANISH) ......................................................................................................... XI ABBREVIATIONS ........................................................................................................................ XV 1.

INTRODUCTION...................................................................................................................... 1

2.

BACKGROUND ........................................................................................................................ 2

2.1

Morphology and life cycle of Taenia solium, Linnaeus 1758 ............................................................................... 2

2.2

Distribution and burden ......................................................................................................................................... 3

2.3

Risk factors and transmission ................................................................................................................................ 6 Taeniosis .......................................................................................................................................................... 6 Porcine cysticercosis........................................................................................................................................ 6 Human cysticercosis ........................................................................................................................................ 8

2.4

Clinical symptoms and diagnostics ........................................................................................................................ 9 Taeniosis .......................................................................................................................................................... 9 Porcine cysticercosis...................................................................................................................................... 11 Human cysticercosis ...................................................................................................................................... 14

2.5

Intervention tools and control .............................................................................................................................. 16 Preventive chemotherapy of humans ............................................................................................................. 16 Improved pig production ............................................................................................................................... 17 Pig vaccination .............................................................................................................................................. 18 Pig treatment .................................................................................................................................................. 18 Meat inspection and processing ..................................................................................................................... 19 Health education and sanitation ..................................................................................................................... 19 Combined intervention tools.......................................................................................................................... 21 Control of Taenia solium ............................................................................................................................... 23

2.6

Transmission modelling to assess interventions for the control of Taenia solium ........................................... 24

2.7

Justification............................................................................................................................................................ 25

3.

OWN INVESTIGATIONS ...................................................................................................... 27

3.1

Objectives............................................................................................................................................................... 27

3.2

Materials and methods - limitations .................................................................................................................... 28 Laboratory examinations ............................................................................................................................... 29

3.3

Results and discussion........................................................................................................................................... 30

3.3.1

Distribution of Taenia solium and the co-distribution with schistosomiasis in Africa (Paper I) ................... 30

3.3.2

Temporal fluctuations and risk factors of porcine cysticercosis (Paper II & III)........................................... 32

3.3.3

Presence of Taenia hydatigena - implications for diagnosis of porcine cysticercosis (Paper IV) ................. 35

3.3.4

School-based praziquantel MDA – effect on taeniosis and porcine cysticercosis (Paper V & VI) ............... 36

3.3.5

Modelling transmission of Taenia solium based on data from an endemic area (Paper VII) ........................ 41

4.

CONCLUSIONS AND PERSPECTIVES ............................................................................. 44

5.

REFERENCES ......................................................................................................................... 48

6.

PUBLICATIONS ..................................................................................................................... 70

6.1

Paper I .................................................................................................................................................................... 70

6.2

Paper II .................................................................................................................................................................. 85

6.3

Paper III ................................................................................................................................................................. 93

6.4

Paper IV ................................................................................................................................................................. 99

6.5

Paper V ................................................................................................................................................................ 108

6.6

Paper VI ............................................................................................................................................................... 115

6.7

Paper VII ............................................................................................................................................................. 123

Preface The work presented in this thesis was undertaken from 2012 to 2016 at the Section for Parasitology and Aquatic diseases, Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, the Faculty of Agriculture, Sokoine University of Agriculture, Tanzania, the Tanzania Livestock Research Institute, Tanzania, the Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Belgium, and at the Department of Biomedical Sciences, Institute of Tropical Medicine, Belgium. The work presented involved collaboration with colleagues from the Faculty of Medicine, School of Public Health, Imperial College London, United Kingdom, the School of Life Sciences, University of KwaZulu-Natal, South Africa, the Institute of Health and Society, Université catholique de Louvain, Belgium, the Department of Public Health and Surveillance, Scientific Institute of Public Health (WIV-ISP), Belgium, the Section of Epidemiology, Vetsuisse Faculty, University of Zurich, Switzerland, the Department of Veterinary Medicine and Public Health, Sokoine University of Agriculture, Tanzania, and Bora Professional Consultancy Services, Tanzania. The PhD was funded by a scholarship from the Faculty of Health and Medical Sciences, University of Copenhagen, the Bill and Melinda Gates Foundation (ICTC-project), the Danish International Development Agency (SLIPP-project), and CYSTINET: European Network on taeniosis/cysticercosis (COST ACTION TD1302).

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During my time as a PhD student I co-authored the following papers not included in this thesis: 

Ertel, R.L., Braae, U.C., Ngowi, H.A., Johansen, M.V., 2017. Assessment of a computerbased Taenia solium health education tool 'The Vicious Worm' on knowledge uptake among professionals and their attitudes towards the program. Acta Trop. 165, 240-245. http://dx.doi.org/10.1016/j.actatropica.2015.10.022



Gabriël, S., Dorny, P., Mwape, K.E., Trevisan, C., Braae, U.C., Magnussen, P., Thys, S., Bulaya, C., Phiri, I.K., Sikasunge, C.S., Makungu, C., Afonso, S., Nicolau, Q., Johansen, M.V., 2017. Control of Taenia solium taeniasis/cysticercosis: the best way forward for subSaharan Africa? Acta Trop. 165, 252-260. http://dx.doi.org/10.1016/j.actatropica.2016.04.010



Johansen, M.V., Trevisan, C., Braae, U.C., Magnussen, P., Ertel, R.L., Mejer, H., Saarnak, C.F.L., 2014. The Vicious Worm: a computer-based Taenia solium education tool. Trends Parasitol. 30, 372-374. http://dx.doi.org/10.1016/j.pt.2014.06.003



Johansen, M.V., Trevisan, C., Gabriel, S., Magnussen, P., Braae, U.C., 2016. Are we ready for Taenia solium cysticercosis elimination in sub-Saharan Africa? Parasitology, 1-6. http://dx.doi.org/10.1017/S0031182016000500

ii

This thesis is based on the following seven papers: 1) Braae, U.C., Saarnak, C., Mukaratirwa, S., Devleesschauwer, B., Magnussen, P., Johansen, M.V., 2015. Taenia solium taeniosis/cysticercosis and the co-distribution with schistosomiasis in Africa. Parasit. Vectors. 8, 323. http://dx.doi.org/10.1186/s13071-0150938-7 2) Braae, U.C., Magnussen, P., Lekule, F., Harrison, W., Johansen, M.V., 2014. Temporal fluctuations in the sero-prevalence of Taenia solium cysticercosis in pigs in Mbeya Region, Tanzania. Parasit. Vectors. 7, 574. http://dx.doi.org/10.1186/s13071-014-0574-7 3) Braae, U.C., Harrison, W., Lekule, F., Magnussen, P., Johansen, M.V., 2015. Feedstuff and poor latrines may put pigs at risk of cysticercosis — A case-control study. Vet. Parasitol. 214, 187-191. http://dx.doi.org/10.1016/j.vetpar.2015.08.009 4) Braae, U.C., Kabululu, M., Nørmark, M.E., Nejsum, P., Ngowi, H.A., Johansen, M.V., 2015. Taenia hydatigena cysticercosis in slaughtered pigs, goats, and sheep in Tanzania. Trop. Anim. Health Pro. 47, 1523-1530. http://dx.doi.org/10.1007/s11250-015-0892-6 5) Braae, U.C., Magnussen, P., Ndawi, B., Harrison, W., Lekule, F., Johansen, M.V., 2017. Effect of repeated mass drug administration with praziquantel and track and treat of taeniosis cases on the prevalence of taeniosis in Taenia solium endemic rural communities of Tanzania. Acta Trop. 165, 246-251. http://dx.doi.org/10.1016/j.actatropica.2015.10.012 6) Braae, U.C., Magnussen, P., Harrison, W., Lekule, F., Ndawi, B., Johansen, M.V., 2016. Effect of national schistosomiasis control programme on Taenia solium taeniosis and porcine cysticercosis in rural communities of Tanzania. Parasite Epidemiol. Control. 1, 245251. http://dx.doi.org/10.1016/j.parepi.2016.08.004 7) Braae, U.C., Devleesschauwer, B., Gabriel, S., Dorny, P., Speybroeck, N., Magnussen, P., Torgerson, P., Johansen, M.V., 2016. cystiSim – an agent-based model for Taenia solium transmission and control. Plos Negl. Trop. Dis. 10, e0005184. http://dx.doi.org/10.1371/journal.pntd.0005184

Uffe Christian Braae Copenhagen, November 2016

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Acknowledgements There are many people that have contributed both directly and indirectly to the work presented in this thesis and the work I have carried out parallel to my PhD. There is unfortunately not enough space in such a thesis to add all the flattering words that you all so rightfully deserve, so please forgive me for being both brief and negligent. First and foremost, I would like to extend my sincere gratitude to my two supervisors Maria Vang Johansen and Pascal Magnussen, without whom this work would not have made a significant impact. Maria, it seems like a lifetime ago since I first stepped into your office with the ambition of doing a PhD. I could not have imagined nor hoped for a better educational journey than the one I embarked on the very first day I set foot in your office. Not only have you been my Lonely Planet, but you have also been a great source of inspiration and support. I have loved (almost) every bit of the journey, but like all journeys it must at some point come to an end. Thank you so much for a fantastic experience and helping me reach my goals! Thanks for always believing in me, for all the countless opportunities to do great and interesting research, and not least, for always caring. I am grateful all in all for the help you have given me to make a difference to the people this work is all about – the farmers in Mbeya, Tanzania. As I soon (hopefully) set out on the next journey on my own, I hope and believe that we in the near future once again will commence a new journey together. Pascal, thank you so much for your guidance and for always listening when I have been in need to discuss issues both large and small. From day one you have guided and supported me, and always found time to give constructive critique and engage in inspirational discussions. I will always treasure the good moments we have had together over the past years, particularly outside the work place. I hope we will continue to meet for a beer or two in the future and I hope that this PhD doesn’t mark the end, but merely the beginning of more fantastic work together over the years to come. I have truly been privileged to have been supervised by two individuals, who are not only great researchers, but fantastic and caring people. Once again, thanks a lot Maria and Pascal.

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I have on several occasions spent many months in Tanzania during my studies, and on each occasion it has felt like coming home. Simon Rwegayura my warmest thanks go to you and your family for opening up your home, for always making me feel welcome and like a member of the family. Feliciana, thank you for the many wonderful home cooked meals; there really is nothing better after coming home from the field in Mbeya. I would also like to thank Professor Faustin Lekule for facilitating my work, always welcoming me to Tanzania, and the many years of good collaboration we have had. I have enjoyed every moment in the field, mostly because I was always surrounded by amazing people who showed incredible spirit. Thanks Mwemezi Kabululu (KB), Albert Manyesela (Cheche), Felician Makiriye, Zhungo Mwaselela, Asukanie Kamanda, Eliakunda Kimbi, and Dr Benedict Ndawi for your fantastic company, and for all your hard work under very tough conditions. This PhD thesis would not have existed without you! I would like to take this opportunity to once again thank all my co-authors for their collaboration, contributions, and constructive critique, thanks Wendy Harrison, Brecht Devleesschauwer, Sarah Gabriël, Pierre Dorny, Niko Speybroeck, Paul Torgerson, Peter Nejsum, Helena Aminel Ngowi, Christopher F. L. Saarnak, Michelle Elisabeth Nørmark, and Samson Mukaratirwa. Also, thanks to all my other collaborators and to CYSTINET for facilitating my work and contributing to an interesting research community. At the University of Copenhagen I would like to thank everybody at the Section for Parasitology and Aquatic Diseases (PAD) for making my place of work, a fantastic one – better colleagues are hard to come by. Although, I have not always been around, I am especially glad for the many good times I have had with my colleagues outside the work place. Thanks Olivier Desrues, Tina Vicky Alstrup Hansen, Andrew Richard Williams, Eline Palm Hansen, Sundar Thapa, Anna M. O. Kildemoes, Laura J. Myhill, Lise-Lotte Christiansen, Annette Olsen, Helena Mejer, and Birgitte J. Vennervald for making PAD more than just a place of work. A special thanks to the PARZOO research group for always making my Friday mornings interesting and focused – great ideas arise in the midst of fruitful discussions. I’m grateful to Kirsten Grønlund Andersen for always lending a helping hand with practical issues and for setting me straight when needed . I would also like to show my appreciation to the Faculty of Health and Medical Sciences, University of Copenhagen for granting me a PhD scholarship. To my family here in Denmark and Belgium, whom I can always trust and depend on to be there for me, thank you! I have reached this far thanks to you. Thanks to all my friends that have been v

committed to listening to me talk about parasites and PhD student life over the past years. I’m sure it has been extremely interesting for you to listen to me speak for hours ;). To Pia, although you haven’t been there for the entire journey, you have been my light and my support through the most important, hardest, and darkest part of my PhD journey. Thanks for being there for me. You have through your support and love made this work great. I apologise for the times you had to endure me ranting on about my PhD work. I could not have succeeded in doing this work without my officemate Chiara Trevisan. Honestly, I probably could have ;), but it would have been nowhere near as much fun without you Chiara. You have always been there for me! Thanks for being you – bringing a little chaos into my life, all the dinners, the travels, the meetings and conferences, and the once or twice (can’t have been more than that) we have gone to A-vej for a quiet Friday beer. It truly is the end of an era!

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Summary Taenia solium taeniosis/cysticercosis is a global health problem, especially in low-income countries in Asia, Latin America, and sub-Saharan Africa. This zoonotic parasite was more than two decades ago declared eradicable, but so far control has not been achieved in any low- or middle-income country. This illustrates that the diseases caused by this parasite are linked to poverty and most often affect only the poorest of the poor. The largest disease burden caused by T. solium is cysticercosis in humans, which is classified as a Neglected Tropical Disease. In sub-Saharan Africa only a few small scale and short-term intervention studies against T. solium have been carried out, whereas actual attempts for control have not been trialled. Challenges are missing links between human and animal health, complicated by absence of collaboration between health and livestock sectors at the ministerial level, in addition to, lack of international funding focusing on controlling this problem. However, to be sustainable control initiatives cannot solely depend on international donors in the long run. Despite T. solium getting more international attention, there are gaps in our understanding of how and when transmission of this parasite occurs, and in sub-Saharan Africa little is known about the distribution, and therefore the burden. Control of other parasitic diseases such as schistosomiasis through praziquantel treatment, also efficacious against taeniosis, is currently on-going in many African countries. The properties of praziquantel enables integrated control, but so far options for integrating control of T. solium within existing programmes have not been assessed. Therefore, the objective of the present project was to describe the epidemiology and potential impact on control of T. solium, by exploring transmission dynamics, assessing integration of control with schistosomiasis control, and developing a transmission model to evaluate intervention options, thereby contributing towards future design and implementation of feasible and sustainable control efforts against T. solium in sub-Saharan Africa. The distribution of T. solium was mapped at district level in Africa, based on published literature and reports from 1985 to 2014 as presented in Paper I. By combining the district level distribution with known presence of schistosomiasis the extent of co-distribution between T. solium taeniosis/cysticercosis and schistosomiasis was investigated. Taenia solium was present in 31 countries and the distribution was determined at district level in 476 districts. Of these districts schistosomiasis was co-distributed in 124 districts in 17 countries. These results both illustrate that T. solium is likely to be highly underreported, and that co-distribution with schistosomiasis is not uncommon, which emphasises the need to explore options for integrated control. vii

In Paper II we investigated temporal fluctuations in sero-prevalence of porcine cysticercosis measured by Ag-ELISA at three time points. The study showed that the fluctuations in prevalence was not associated to confinement of pigs and that confined pigs were just as likely to be infected as free-range and semi-confined pigs. This led to the hypothesis that the transmission of T. solium in confined pigs occurs within the pig pens through contaminated feed or water. To explore the hypothesis of contaminated feed, the association of persistent or multiple infections in pigs with different feedstuff and latrine type was investigated using a case-control design at herd level in Paper III. The study showed that missing or poor latrines at the household and feeding potato peels to pigs were risk factors for porcine cysticercosis. The results indicate a contamination of the environment with Taenia eggs during the study period. This underlines the importance of including pig management in control efforts and that the lack of knowledge about the mechanism and duration of contamination should be addressed in order to make control efforts more realistic and sustainable. The finding that confinement of pigs, under the study settings, had no effect on reducing the risk of infection with porcine cysticercosis compared to free roaming pigs was surprising. Difference in prevalence of porcine cysticercosis under different types of management systems, such as pen type, has been seen before in the area, and corroborated here. The association of using specific feed, such as potato by-products, and infection with cysticercosis in pigs has not been reported before. These results should be taken into consideration within control programmes containing a pig production component. Measuring impact of control programmes, if evaluated by changes in porcine cysticercosis prevalence, could be complicated by the occurrence of temporal fluctuations in prevalence. The presence of Taenia hydatigena has been described in pigs only once before in Tanzania. Paper IV describes two slaughter slab surveys investigating the prevalence of T. hydatigena in pigs, goats, and sheep. The surveys revealed the presence of T. hydatigena in Mbeya Region, and found a relative high prevalence (6.6%) in pigs. The prevalence of T. hydatigena can be an important factor when measuring the effect of intervention tools against T. solium in pigs if measurements are based on Ag-ELISA due to the cross-reaction between T. hydatigena and T. solium. Paper V describes the first assessment of the short-term effect on taeniosis of repeated schoolbased praziquantel mass drug administration combined with ‘track-and-treat’ of taeniosis cases in Africa. Based on copro-Ag prevalence the study illustrated that annual treatment of school-aged viii

children was significantly better compared to a single treatment in reducing prevalence of taeniosis in the target group. Short-term, there was no effect of the intervention on prevalence of taeniosis in the non-target population (adults). Paper VI presents the effect of national schistosomiasis control programme interventions on both taeniosis and porcine cysticercosis when followed over a four year period. Annual school-based praziquantel mass drug administration proved better compared to biennial school-based praziquantel mass drug administration by indicating effects not only on the target population, but also reduced the prevalence of taeniosis within the adult population and the prevalence of porcine cysticercosis in the intermediate host population. The study also highlighted that cultural or behavioural differences between districts should be considered if targeted preventive chemotherapy is planned, as males were found to be more at risk in one district compared to the other, and that risk increased with age. A mathematical model has been developed to explore transmission dynamics of T. solium. As the existing model is based on data from different geographical locations in Latin America, lacks age structures, and requires user input of degree of transmission reduction, the computational model cystiSim was developed. Paper VII describes the capabilities and limitations of cystiSim, the first agent-based model for transmission and control of T. solium to evaluate intervention approaches. The model simulations showed that the most robust and likely intervention strategy to make and impact on prevalence was a combination of mass drug administration to humans combined with treatment and vaccination of pigs. cystiSim does however predict that elimination of the parasite is possible using a single intervention approach, although the time frame is longer and results are more susceptible to changes in coverage. In conclusion, no national initiatives for control of T. solium currently exist in Africa despite that the distribution of T. solium covers most of sub-Saharan Africa on a national level. For many areas, especially at district level, limited information is currently available about T. solium presence. Detailed information about the distribution of T. solium is necessary to estimate the burden of the parasite and to make informed decisions on whether control initiatives can be justified. Codistribution of T. solium taeniosis/cysticercosis and schistosomiasis does occur, and in Tanzania the National Schistosomiasis Control Programme seemed to have an effect on taeniosis prevalence, and in areas with annual treatment, also on porcine cysticercosis. This could call for the integration of T. solium control in already existing programmes. However, cost-effect and cost-benefit analyses will have to be made to investigate whether new areas should be included within control programmes ix

based on T. solium presence. Praziquantel mass drug administration was unable to break the parasite life cycle in the time frame investigated, and therefore presumably unsuitable as a stand-alone tool. cystiSim did however predict that community-wide mass drug administration could break the life cycle, if the duration of the programme was extended to more than 25 years, which would add extra requirements for a control programme in terms of securing long-term funding. cystiSim predicted the best-bet option for control to be a One Health approach targeting both the definitive host through mass drug administration, and the intermediate host through mass treatment and vaccination programmes. Not included in the simulations, but likely to make significant difference for transmission control and the degree of success of a control programme, is the implementation of effective meat inspection and inclusion of health education with focus on sanitation, hygiene, and health information. If cystiSim proves valid, then decision makers will have a tool capable of guiding them in what is needed in terms of interventions to reach the levels of control they desire.

x

Sammendrag (Danish) Taenia solium taeniose/cysticercose er et globalt sundhedsproblem, især i lavindkomstlande i Asien, Latinamerika og Afrika. Denne zoonotiske parasit (svinetintebændelorm) blev for mere end to årtier siden erklæret mulig at udrydde, men indtil videre er kontrol eller elimination ikke opnået i hverken lav- eller mellem-indkomstlande. Sygdomme forårsaget af denne parasit er knyttet til fattigdom og rammer derfor de fattigste befolkningsgrupper. De sværeste sygdomstilfælde i mennesker skyldes at svinetintebændelorm danner tinter i hjernen, kaldet neurocysticercose. Til trods for øget international opmærksomhed, og at T. solium cysticercose er klassificeret som en overset tropisk sygdom (Neglected Tropical Disease), er der i Afrika kun udført meget få mindre og kortsigtede interventionsstudier mod T. solium, og egentlige forsøg på kontrol er ikke blevet afprøvet. En af udfordringerne, på grund af det zoonotiske aspekt, er den manglende forbindelse mellem menneskers og dyrs sundhed, hvilket yderligere kompliceres af manglende samarbejde mellem sundheds- og landbrugssektorer på ministerielt plan, kombineret med manglende international finansiering med fokus på at kontrollere denne parasit. For at opnå bæredygtig kontrol kan kontrolprogrammer dog ikke, på sigt fastholde et afhængighedsforhold til internationale donorer, men må baseres på at de enkelte lande afsætter nationale ressourcer til bekæmpelse. Der er huller i vores forståelse af, hvor, hvordan og hvornår transmission af denne parasit finder sted. Især i Afrika vides meget lidt om den egentlige geografiske udbredelse, og derfor også byrden, såvel sygdommæssige og økonomisk, af T. solium. Praziquantel massebehandling bruges til kontrol af andre parasitiske sygdomme såsom schistosomiasis (sneglefeber) i mange afrikanske lande. Da praziquantel også er effektivt mod taeniose muliggør det integreret kontrol af T. solium og andre ormesygdomme. Tidligere er integration af T. solium kontrol i eksisterende programmer dog ikke blevet vurderet. Formålet med dette projekt var at få indsigt i epidemiologien og kontrol af T. solium, ved at udforske transmissionsdynamik, vurdere mulighederne for integrationen af kontrol med eksisterende schistosomiasis kontrol, og udvikle en transmissionsmodel til evaluering af interventionsmuligheder, og derved bidrage til fremtidige udformninger og implementering af en bæredygtig kontrolindsats mod T. solium i Afrika syd for Sahara. For Afrika blev udbredelsen af T. solium kortlagt på distriktsniveau, baseret på tilgængelig litteratur og rapporter fra 1985 til 2014 som er præsenteret i Artikel I. Ved at kombinere udbredelsen på distriktsplan med forekomsten af schistosomiasis kunne begges sygdommes udbredelse og overlap kortlægges. Taenia solium blev i perioden rapporteret i 476 distrikter i 31 lande. Af disse 476 xi

distrikter var schistosomiasis reporteret i 124 distrikter i 17 lande. Disse resultater illustrerer at T. solium sandsynligvis er underrapporteret, og at samtidig forekomst med schistosomiasis ikke er ualmindeligt. Dette understreger behovet for at undersøge mulighederne for integreret kontrol. Artikel II beskriver temporale (tidsmæssige) svingninger i seroprævalens af cysticercose i svin målt ved Ag-ELISA inden for 16 måneder. Undersøgelsen viste, at udsvingene i prævalens ikke var forbundet til indespærring af svin, og at indhegnede svin var lige så tilbøjelige til at blive smittet som fritgående og delvist fritgående svin. Dette førte til hypotesen at transmissionen af T. solium blandt svin på stald sker inden for indhegningen gennem forurenet foder eller vand. For at udforske hypotesen om forurenet foder, blev associationen mellem vedvarende og gentagne infektioner i svin med forskellige foderstoffer og latrintyper undersøgt ved hjælp af et case-kontrol design. Resultaterne af undersøgelsen beskrives i Artikel III. Undersøgelsen viste, at manglende eller dårlige latriner i husstanden og fodring af svin med kartoffelskræller var risikofaktorer for cysticercose i svinebesætninger. Resultaterne indikerer en forurening af miljøet med Taenia æg i undersøgelsesperioden. Dette understreger vigtigheden af øget fokus på forbedret svineproduktion som led i en kontrolindsats, og at manglende viden omkring varighed af forureningen skal undersøges for at gøre en potentiel kontrolindsats mere realistisk og bæredygtig. At indhegning af svin i denne undersøgelse ingen indflydelse havde på risikoen for cysticercose i svin sammenholdt mod fritgående svin var overraskende. Forskel i forekomsten af svin med cysticercose under forskellige typer af produktionssystemer, såsom sti-type er før observeret i området, og bekræftet her. Resultatet af sammenhængen mellem anvendelsen af kartoffelskræller som foderstof og infektion med cysticercose i svin, bør tages i betragtning i eventuelle kontrolprogrammer der indeholder en svineproduktionskomponent. Effektestimeringen af kontrolprogrammer kan kompliceres af tidsmæssige svingninger i prævalens af cysticercose i svin, hvis effekten vurderes ud fra ændringer i prævalens. Tilstedeværelsen af Taenia hydatigena er kun en gang tidligere blevet beskrevet i svin i Tanzania. Artikel IV beskriver en undersøgelse af forekomsten af T. hydatigena i svin, geder og får slagtet i Mbeya distriktet i Tanzania. Undersøgelserne afslørede tilstedeværelsen T. hydatigena i Mbeya, og identificerede en relativ høj prævalens (6,6%) hos svin. Forekomsten af T. hydatigena kan være en vigtig parameter ved effektestimering af interventionsredskaber mod T. solium hos svin, hvis estimeringen er baseret på Ag-ELISA fordi forekomsten af T. hydatigena og T. solium ikke kan differentieres. xii

Artikel V beskriver den første vurdering i Afrika af den kortsigtede effekt på taeniose af gentagne skolebaserede praziquantel massebehandlinger kombineret med ”track-and-treat" af taeniose tilfælde. Baseret på copro-Ag prævalens viste undersøgelsen at den årlige behandling af skolebørn var signifikant bedre i forhold til en enkelt behandling, til at reducere forekomsten af taeniose i målgruppen. Kortsigtet var der ingen effekt af interventionen på forekomsten af taeniose udenfor målgruppen (voksne). Artikel VI beskriver effekten af Nationale Schistosomiasis Kontrolprogram i Tanzania på både taeniose og cysticercose i svin over en fireårig periode. Årlig skolebaseret praziquantel

massebehandling

viste

sig

bedre

i

forhold

til

skolebaseret

praziquantel

massebehandlinger hvert andet år ved at have effekt ikke kun på målgruppen, men også ved at reducere forekomsten af taeniose i den voksne befolkningsgruppe og reducere forekomsten af cysticercose i svin. Undersøgelsen fremhævede også at kulturelle eller adfærdsmæssige forskelle mellem distrikter bør overvejes, hvis målrettet forebyggende kemoterapi er planlagt, da mænd viste sig at have højere risiko i det ene af de involverede distrikter end det andet og at risikoen generelt steg med alderen. En matematisk model er tidligere blevet udviklet til at undersøge transmissionsdynamikken af T. solium. Modellen byggede på data fra forskellige geografiske områder i Latinamerika, men mangler aldersstrukturer, og forudsætter graden af transmissionsreduktion fra brugeren. For at imødekomme dette, blev den agentbaseret model cystiSim udviklet. Artikel VII beskriver de muligheder og begrænsninger cystiSim har med hensyn til at vurdere forskellige interventioners effekt på kontrol af T. solium. Modelsimuleringerne viste, at den mest robuste og sandsynlige strategi til at påvirke forekomsten af T. solium var en kombination af massebehandling af mennesker kombineret med massebehandling og vaccination af svin. Dog estimerede cystiSim at eliminering af parasitten var mulig ved anvendelse af en enkelt interventionsstrategi, selvom tidsrammen var længere og resultaterne mere påvirkelige over for ændringer i dækningsgraden af interventionen. I øjeblikket findes ingen nationale initiativer til kontrol af T. solium i Afrika til trods for at udbredelsen af T. solium omfatter de fleste lande i Afrika syd for Sahara. For mange områder, især på distriktsniveau, er information om parasittens tilstedeværelse meget sparsom. Detaljerede oplysninger om den geografiske udbredelsen af T. solium er nødvendig for at bedømme betydningen af parasitten og træffe kvalificerede beslutninger om hvorvidt kontrolinitiativer kan begrundes. Co-distribution af T. solium taeniose/cysticercose og schistosomiasis forekommer hyppigt, og i Tanzania synes det nationale schistosomiasis kontrolprogram at have en effekt på xiii

forekomsten af taeniose og i områder med en årlig behandling, en effekt på cysticercose i svin. Derfor bør integration af T. solium kontrol i allerede eksisterende programmer vurderes yderligere. Cost-effekt og cost-benefit analyser bør udføres for at undersøge, om nye områder bør indgå i kontrolprogrammer baseret på T. solium tilstedeværelse. Massebehandling med praziquantel var ikke i stand til at bryde parasittens livscyklus i vores undersøgelses tidsramme og er derfor formentligt utilstrækkeligt som et enkeltstående værktøj. cystiSim forudsagde dog, at massebehandling af hele befolkningen kan bryde livscyklus, hvis tidsrammen for programmet forlænges, hvilket tillægger ekstra krav til langsigt finansiering af programmet. cystiSim forudsagde at den bedste mulighed for kontrol var en One Health tilgang, rettet mod både slutværten gennem massebehandling, og mellemværten gennem massebehandling og vaccination. Ikke inkluderet i simuleringerne, men formentlig væsentlig for transmissionskontrol og graden af succes af et kontrolprogram

er

implementeringen

af

effektiv

kødkontrol

og

inddragelse

af

sundhedsundervisningen med fokus på sanitet, hygiejne og sundhedsoplysning. Bliver cystiSim succesfuldt valideret, vil beslutningstagere få et redskab i cystiSim der er i stand til at vejlede dem i hvilke tiltag og frekvensen heraf, der er behov for at opnå et ønsket niveau af kontrol.

xiv

Abbreviations Ag

Antigen

ASF

African swine fever

CLTS

Community-Led Total Sanitation

CSF

Cerebrospinal fluid

CT

Computerized tomography

DALY

Disability-Adjusted Life Year

EITB

Enzyme-linked immuneelectrotransfer blot

ELISA

Enzyme-linked immunosorbent assay

GNTD

Global Neglected Tropical Disease Database

ITFDE

International Task Force for Disease Eradication

MDA

Mass drug administration

MRI

Magnetic resonance imaging

NCC

Neurocysticercosis

NSCP

National Schistosomiasis Control Programme

NTD

Neglected Tropical Diseases

OD

Optical density

OIE

World Organisation for Animal Health

RFLP

Restriction fragment length polymorphism

WASH

Water, sanitation, and hygiene

WHO

World Health Organization

YLD

Years lived with disability

YLL

Years of life lost due to premature mortality

xv

1. Introduction Taenia solium taeniosis/cysticercosis was declared eradicable by the International Task Force for Disease Eradication (ITFDE) in 1993, and has in the past decades been recognised as a serious agricultural and public health problem in sub-Saharan Africa (Karesh et al., 2012; Phiri et al., 2003), Asia (Rajshekhar et al., 2003), and Latin America (Garcia et al., 2003b). More recently T. solium was identified as the most important foodborne parasite globally (FAO and WHO, 2014). In 2010, the World Health Organization (WHO) included T. solium cysticercosis as one of major Neglected Tropical Diseases (NTD) and recommended human mass chemotherapy as the primary intervention strategy against taeniosis (WHO, 2010). Still, no large scale control programme for taeniosis has been implemented in sub-Saharan Africa to date, nor has the effect of mass drug administration (MDA) or integrating T. solium control with other existing control programmes e.g. schistosomiasis control, been fully assessed. The World Health Assembly passed the WHA66.12 resolution in 2013 with the aim of eliminating T. solium as a public health problem, and WHO is therefore supporting activities in selected countries with the goal of scaling up interventions by 2020 (WHO, 2015a). Porcine cysticercosis has hampered the sub-Saharan pig production, significantly reducing the market value of infected pork, resulting in economic losses for farmers with infected pigs (Carabin et al., 2006). Due to high population growth, sub-Saharan Africa is faced with several challenges such as food insecurity and disease burdens. Pigs are highly proliferative and have the ability of converting otherwise wasted resources into high dietary protein, and are therefore, of importance in alleviating some of these problems. The demand for pork is projected to drastically rise in lowincome countries within the next decades (Delgado, 2003), and with it, the potential emergence of T. solium taeniosis/cysticercosis. Although theoretically controllable (Kyvsgaard et al., 2007) and declared eradicable, T. solium cysticercosis remains a neglected disease due to lack of information about its burden, transmission, and validation of simple intervention packages (WHO, 2010). No national T. solium specific control strategies are currently in place in sub-Saharan Africa. To succeed, a control strategy will be dependent on both local and political acceptance, commitment, and engagement (Gabriel et al., 2017).

1

2. Background 2.1 Morphology and life cycle of Taenia solium, Linnaeus 1758 Cestodes belong in the phylum Platyhelminthes under the animal kingdom. Humans can host adult species of three cestodes from the genus Taenia: T. solium, T. saginata, and T. asiatica. Taenia saginata and T. asiatica are genetically closer related compared to T. solium (Bowles and McManus, 1994; de Queiroz and Alkire, 1998). Two genotypes of T. solium have been described, one from Asia and one from Africa/Americas (Bowles and McManus, 1994; Ito et al., 2003b; Nakao et al., 2002; Singh et al., 2016). Although, both genotypes have been found in Madagascar (Michelet et al., 2010), it is currently unknown if the Asian genotype is present in other African countries. Morphologically T. solium has a scolex with a double row of hooks and four suckers. The strobila is made up of proglottids each containing 50,000 to 60,000 eggs when gravid (Flisser, 1994). Taenia solium is transmitted between humans and pigs (Figure 1). Humans are the definitive host and infected with the adult stage of the tapeworm (taeniosis) by ingesting raw or improperly cooked pork containing cysticerci, the larval stage, which then develops in the small intestines of the human host. Eggs are excreted approximately after two months, yet the length of survival of the adult tapeworm within the host remains unknown (Pawlowski, 2002). Studies have indicated that the tapeworm is unlikely to survive 25 to 30 years within the human host as anecdotally mentioned in literature (Allan et al., 1996; Garcia et al., 2003a; Mwape et al., 2012), instead the lifespan is probably between one to three years (Lightowlers, 2010). Typically humans with T. solium taeniosis harbour only a single tapeworm (Pawlowski, 2002), but infections with multiple worms, or even multiple species, have been reported (Jeri et al., 2004; Wandra et al., 2011). Proglottids or eggs are immotile and excreted intermittingly by tapeworm carriers, thereby contaminating the environment, if faeces are disposed of improperly. Both humans and pigs can be infected with the larval stage of the tapeworm (cysticercosis) by ingesting tapeworm proglottids or eggs excreted from human tapeworm carriers. Human cysticercosis can manifest as neurocysticercosis (NCC) characterised by cysts located within the central nervous system causing severe neurological disorders (Garcia et al., 2003b), but this plays no role in sustaining the transmission of T. solium. Human cysticercosis can also manifest as ocular cysticercosis, where cysts are lodged in the eye (Rahalkar et al., 2000), or as subcutaneous cysticercosis characterised by the presence of subcutaneous nodules, mostly seen in Asia and to some degree in Africa, but rarely in Latin America (Garcia et al., 2003b). However, the occurrence and comparison of subcutaneous nodules on regional levels have not been systematically assessed. 2

Figure 1: Life cycle of Taenia solium - adapted from (Braae et al., 2017).

2.2 Distribution and burden Taenia solium is distributed globally (Figure 2), but mostly associated with low-income countries where pigs are kept, and is presumed widely distributed in Latin America (Garcia et al., 2003b), Africa (Braae et al., 2015d; Phiri et al., 2003; Zoli et al., 2003), and Asia (Rajshekhar et al., 2003). Taenia solium is also suspected to be present in parts of Europe (Devleesschauwer et al., 2017). In sub-Saharan Africa the distribution is not well documented and the parasite is suspected to be grossly underreported, but has recently been documented in 31 countries (Braae et al., 2015d) (see section 3.3.1). Taenia solium has been shown to cluster (Garcia et al., 2003a; Ngowi et al., 2010; Raghava et al., 2010). In Peru, porcine cysticercosis was shown to cluster around households with a tapeworm carrier (Garcia et al., 2003a).

3

Figure 2: Global distribution of Taenia solium, 2015 (WHO, 2016)

The two main (disease and economic) burdens of T. solium are neurological complications (NCC) detrimental to human health and monetary loss to pig production due to porcine cysticercosis. The human disease burden is traditionally measured using the metric Disability-Adjusted Life Year (DALY), where one DALY is equivalent to one year of healthy life lost (Murray, 1994). Developed for the Global Burden of Disease study (Murray and Lopez, 1997), DALY combines years of life lost due to premature mortality (YLL) and years of life lost due to time lived with disability (YLD), thus DALY = YLL + YLD. Human cysticercosis was recently estimated to be the foodborne parasite with the highest DALY globally (Torgerson et al., 2015). WHO estimates that 50 million people are suffering from epilepsy in T. solium endemic countries, and more than one quarter of these cases could result from NCC (Ndimubanzi et al., 2010). However, few of the studies included in the systematic review by Ndimubanzi et al. (2010) originated from sub-Saharan Africa, and differences between regions or genotypes of T. solium might exist. The burden estimations for human cysticercosis are limited by the exclusion of non-specific symptoms potentially caused by NCC, such as severe headache and impaired or lost vision, in addition to cases of mortality due to NCC related epileptic seizures reported as e.g. traffic accidents, severe burn cases, or drowning, which are overlooked. 4

A recent review of human cysticercosis prevalence studies showed that when measured by circulating antigens (Ag), prevalence in Asia was on average 4% whereas the average prevalence in Latin America and Africa was 7% (Coral-Almeida et al., 2015). Prevalence of human cysticercosis in Tanzania has been reported to be up to 17% (Mwanjali et al., 2013) and NCC has been reported to be the likely cause of epilepsy in more than 50% of the people with epilepsy in some endemic areas of Zambia (Mwape et al., 2015). In West Cameroon the health cost due to NCC was estimated to be 10 million EUR in 2009 (Praet et al., 2009). Due to increased migration and travel, human cysticercosis can now be found globally (Burneo et al., 2009; Schantz et al., 1992). In USA from 2004 to 2012, NCC related hospitalisations and associated charges, exceeded the totals for malaria and for all other NTD combined (O'Neal and Flecker, 2015). Globally an unknown number of people have T. solium taeniosis (WHO, 2013), but prevalence in endemic areas are often between 1% to 3% (Coral-Almeida et al., 2015). However, hyper endemic areas exist, and in sub-Saharan Africa taeniosis prevalence of 6% and 12% have been reported (Mwape et al., 2012; Mwape et al., 2013). In Tanzania taeniosis prevalence of 1% to 5% have been reported (Braae et al., 2016b; Braae et al., 2017; Mwanjali et al., 2013). In Asia, taeniosis prevalence can in certain areas reach between 19% and 26% (Okello et al., 2014; Prasad et al., 2007), but is then often inclusive of T. asiatica taeniosis. Taeniosis prevalence in Latin America is comparable to Africa with prevalence of 1% to 4% often reported (Allan et al., 1997; DiazCamacho et al., 1991; Garcia et al., 2003a; Sarti et al., 1992a; Sarti et al., 2000). Prevalence of porcine cysticercosis has in certain areas of sub-Saharan Africa been reported to be as high as 64% (Mwape et al., 2012). Porcine cysticercosis significantly reduces the market value of pork (Atawalna et al., 2015), causing economic losses to farmers and affecting their livelihoods. In China porcine cysticercosis was estimated to cost 121 million USD per year (Ito et al., 2003a). Porcine cysticercosis was estimated to cost 25 million Euro annually for 10 African countries (Zoli et al., 2003). In South Africa, porcine cysticercosis was estimated to cost 5 million USD in the Eastern Cape Province in 2004, and the overall monetary burden of T. solium was between 19 and 34 million USD per year (Carabin et al., 2006). Estimating the societal cost, inclusive of both the DALY and the monetary burden of both the human health and the livestock sector is necessary for estimating the true burden of zoonotic diseases such as T. solium. Trevisan et al. (2017) estimated the societal cost of T. solium in Tanzania per annum anno 2012 to be approximately 9 million USD. However, these estimates are hindered by lack of accurate disease prevalence estimations due to 5

insufficient diagnostic tools, and rough estimates on costs to the livestock sector owing to missing data. As data collection is not standardised across countries, complications comparing figures of burden between countries can occur. Additionally, the limitations of the data should be considered before such comparisons are made. 2.3 Risk factors and transmission Taeniosis Ingesting viable T. solium cysts in infected pork is a prerequisite for acquiring taeniosis. Consuming raw or undercooked pork infected with cysticerci is the major risk factor for becoming infected with taeniosis (Prasad et al., 2007; Sarti et al., 1992b). Cultural preference or poverty forcing individuals to consume infected pork can be perpetuating this behaviour in certain regions (Ito et al., 2013). These risk factors combined with lack of effective meat inspection could also propagate taeniosis in endemic areas (CWGP, 1993). The risk of infection has been shown to increase with age (Braae et al., 2016b; Braae et al., 2017; Prasad et al., 2007), perhaps due to increased consumption of pork, risky behaviour, or an indication that the tapeworm is long lived, which has been anecdotally put forward (Pawlowski and Schultz, 1972). However, other studies have shown age to be of less importance, and the prevalence of taeniosis more evenly distributed within the population, and with approximately 5% of children infected in some areas (Mwape et al., 2012). Mwape et al. (2012) could not associate sex with taeniosis infections in Zambia, but studies from Tanzania have shown males to be more at risk compared to females (Braae et al., 2016b; Braae et al., 2017) (see section 3.3.4). In contrast, females have been reported to be more at risk in Latin America (Allan et al., 1996; Garcia et al., 1998), and this might reflect cultural differences. Incidence of infection is not well studied, but Allan et al. (1997) estimated the incidence to be approximately 10 cases per 1000 people per year within an endemic area of Guatemala. Porcine cysticercosis Pigs become infected with porcine cysticercosis by ingesting T. solium eggs. Several studies have investigated risk factors for porcine cysticercosis. Two risk factors often found are absence or lack of usage of latrines (Boa et al., 2006; Braae et al., 2015a; Jayashi et al., 2012a; Ngowi et al., 2004a; Sarti et al., 1992a), and keeping pigs in a free-range production system (Boa et al., 2006; Komba et al., 2013; Pondja et al., 2010; Pouedet et al., 2002; Sarti et al., 1992a; Sikasunge et al., 2007). However, Braae et al. (2014) found that keeping pigs confined did not reduce the risk of infection, 6

but that feeding potato peels to the pigs was a risk factor (Braae et al., 2015a) (see section 3.3.2). Komba et al. (2013) also found that the source of water could be associated with infection in pigs. The importance of pig management in the transmission of porcine cysticercosis has been further highlighted, by the potential role pig management plays in transmission shown by different pen types, such as elevation of the pen or dirt flooring, which yielded increased risk of infection in pigs (Braae et al., 2015a; Komba et al., 2013). Increasing age of pigs has been reported as a risk factor (Braae et al., 2014; Braae et al., 2016b; Komba et al., 2013; Pondja et al., 2010). This might indicate that pigs do not clear the infection on a regular basis or that older pigs simply eat more feed and are therefore at higher risk if transmission occurs through feedstuff (Braae et al., 2015a). A study on the behaviour of free-range pigs in Mexico showed a hierarchy among pigs resulting in adult pigs more frequently consuming human faeces compared to younger pigs (Copado et al., 2004). When and how often pigs clear an infection is not well understood, but Nguekam et al. (2003a) found that infected piglets were incapable of clearing an infection in the first six months, indicating that pigs infected early in life are unable to clear an infection with T. solium. A study from Mexico suggests that free-range pigs more frequently eat human faeces during the dry season compared to the wet season, perhaps, because other types of food are more readily available during the wet season or that faeces are more often washed away during the wet season (Copado et al., 2004). This could indicate a significant difference in transmission of T. solium to pigs between the two seasons. Porcine cysticercosis can cluster around households with tapeworm carriers, but can also be found at longer distances from households inhabited by tapeworm carriers (Lescano et al., 2007). Although free-range pigs can move several kilometres daily in search of food (Thomas et al., 2013), an environmental vector cannot be excluded in the dispersal of Taenia egg. Dung beetles are expected to be potential mechanical vectors of Taenia eggs because they feed exclusively on faeces. Moreover, dung beetles have been suggested to contribute in the dissemination of Taenia eggs as biological vectors. Taenia eggs can survive in the digestive system of beetles (Gomez-Puerta et al., 2014), and the presence of intestinal nematodes that require dung beetles as their intermediate host, have been associated with both exposure and infection of T. solium in pigs (Arriola et al., 2014). Blowflies have also experimentally been shown to be indirectly capable of transmitting T. hydatigena to pigs by contaminating feedstuff with eggs, either by vomiting or defecating, onto meat fed to pigs (Lawson and Gemmell, 1990). In an experimental infection study, it was shown 7

that the vast majority of eggs did not establish as cysts and pigs that received low doses of eggs had relatively more degenerated cysts (Santamaria et al., 2002), which might indicate a more efficient immune response in pigs with low intensity infections. The fate of non-established eggs was not determined, but it is possible that a proportion of the eggs had passed with the faeces. One study found that pigs consuming proglottids were able to pass eggs in the faeces and infect pen mates, albeit at a lower dose (Gonzalez et al., 2005), but this remains to be validated. In some areas where latrines are unavailable or used infrequently, direct defecation within the pigsty is a common practice (Nguekam et al., 2003b), but more commonly, open defecation would be the leading cause of T. solium transmission to pigs. The degree of T. solium eggs present within the environment of endemic areas due to open defecation is unknown, but levels of eggs should be expected to be high based on the high egg output from people with taeniosis (Flisser, 1994). Taenia eggs have been shown to be extremely difficult to remove from effluent, and were still present in wastewater systems that had other helminth eggs such as Ascaris eggs successfully removed (Verbyla et al., 2013). Few studies have investigated Taenia eggs in the environment such as from soil or water (Diaz-Camacho et al., 1991; Edia-Asuke et al., 2014; Gweba et al., 2010; Keilbach et al., 1989). Unfortunately, none of these studies have quantified the amounts of material investigated and the eggs found were not identified to species level. Taenia saginata eggs are estimated to stay infective for at least five months in soil under temperate conditions (Ilsoe et al., 1990). This relative long survival of eggs could increase the significance of the environment as a parameter for the transmission of porcine cysticercosis and even human cysticercosis. Human cysticercosis Data on the transmission dynamics of human cysticercosis are scarce, and individuals in endemic areas are expected to be highly exposed (based on antibody levels), but few in comparison have active infections based on circulating antigens (Praet et al., 2010; Rodriguez-Hidalgo et al., 2006). Anybody coming into contact with a tapeworm carrier is at risk of contracting cysticercosis, in fact, in New York, USA a Jewish community suffered an outbreak of NCC, believed to have been caused by a housekeeper with taeniosis (Schantz et al., 1992). In India, vegetarians were found to be at risk of cysticercosis from food handlers with taeniosis (Rajshekhar et al., 2003). Major risk factors for human cysticercosis are T. solium taeniosis infection and poor personal hygiene, particularly in combination with each other (Mwape et al., 2012; Sarti et al., 1992b), and poor or insufficient coverage of latrines and sanitation (Secka et al., 2011). Noormahomed et al. (2003) 8

suggested that the main risk factor for human cysticercosis in and around Maputo, Mozambique was the consumption of contaminated fruit and vegetables. Similar results with lack of safe drinking water and consuming raw vegetables as risk factors have been reported in India (Prasad et al., 2011). In Tanzania, risk factors for human cysticercosis were increasing age, washing hands using the ‘dipping method’, and carrying a tapeworm (Mwanjali et al., 2013). The proportion of people with antibodies specific for cysticercosis is generally seen to increase with age (Kanobana et al., 2011; Praet et al., 2010), as are the number of people with asymptomatic cysticercosis (Carabin et al., 2015; Prasad et al., 2011). Kanobana et al. (2011) reported that males were more likely to have cysticercosis compared to females in the Democratic Republic of Congo. In contrast, studies from Latin America have shown that females were more exposed to cysticercosis than males (GarciaNoval et al., 1996; Garcia et al., 1998). These studies indicate that cultural factors play an important role in the transmission of human cysticercosis, and who is at risk can therefore vary between regions. 2.4 Clinical symptoms and diagnostics Taeniosis Taeniosis is characterised by being asymptomatic or yielding mild uncharacteristic symptoms such as abdominal pain, diarrhoea, and nausea. Worm burden in humans can only be established by expulsion with an anthelminthic drug. However, it is presumed that most infected people will only harbour one tapeworm (Wandra et al., 2011). Although, there have been reports of individuals with more than one T. solium tapeworm, as well as cases with people simultaneously infected with more than one Taenia species (Anantaphruti et al., 2007). Taeniosis can be diagnosed by coprology and comparative morphology, immunodiagnostic, or molecular diagnostic approaches, but no single test can guarantee no false negatives (Pawlowski and Schultz, 1972). Coprology in combination with microscopy is highly specific at the genus level as Taenia eggs appear identical, but the sensitivity is low (Praet et al., 2013). Coprology is disadvantaged by the occurrence of false negatives due to immature worms not excreting eggs, and eggs being excreted intermittently by adult worms (Allan et al., 1996; Praet et al., 2013). The latter can be ameliorated by sampling repeatedly over several days (Hall et al., 1981). If whole gravid proglottids or the scolex is expelled in the stool, identification can often be made to species level based on the morphological characteristics. Expulsion of the tapeworm or proglottids can be provoked by 9

administrating anthelminthic treatment, which can be followed by morphological examination as previously explained. However, following successful anthelminthic treatment the scolex cannot be recovered in a majority of cases (Jeri et al., 2004). Also, morphological abnormalities can occur making diagnosis to species level uncertain using microscopy (Rodriguez-Hidalgo et al., 2002). Immunodiagnostic methods are more sensitive compared to microscopy (Deckers and Dorny, 2010). A copro-Ag-ELISA assay based on antibodies obtained from hyperimmune rabbit sera against T. solium tapeworm somatic antigens or excretory-secretory products, has been developed to detect parasite specific antigens to T. solium, T. saginata and T. asiatica in faeces from hosts (Allan et al., 1990). The copro-Ag-ELISA assay has been reported to have high sensitivity (85%) and specificity (92%) (Praet et al., 2013). Antigens are detectable prior to patency, and cease to be detectable one week after successful anthelminthic treatment (Allan et al., 1990). Tembo and Craig (2015) showed using a novel copro-Ag-ELISA, that antigens can be detected steadily 72 hours post infection. But it was also stated that the assay was unreliable and showed mixed results until three months after infection. Wilkins et al. (1999) developed a highly specific and sensitive serologic assay capable of detecting specific T. solium taeniosis antibodies in human serum. Levine et al. (2004) modified the assay by replacing the native proteins with two recombinant antigens (rES33, rES38). The assay was shown in field tests in Peru using an EITB format to have a specificity of 100% and 91% for rES33 and rES38, respectively, and a sensitivity of 97% for rES33 and 98% for rES38, respectively (Levine et al., 2007). A magnetic immunochromatographic test using a particle-labelled detection system to improve detection of antibodies against T. solium taeniosis and cysticercosis has also been developed (Handali et al., 2010), and this point-of-care test can prove important for surveillance in future control programmes. Nevertheless, the assay needs modifications as there are problems with cross-reactions with Echinococcus spp. and Schistosoma Spp. Molecular tools that are species specific with high sensitivity and specificity have also been developed to detect parasite DNA in stool (Chapman et al., 1995; Mayta et al., 2008), but they are expensive, time consuming, and more demanding in terms of sample storage and laboratory requirements, compared to immunodiagnostics. This makes the utilisation of these tools problematic in low-income countries and for large scale programmes. The approaches used to detect DNA material from T. solium are PCR coupled to restriction fragment length polymorphism (RFLP) (González et al., 2000; González et al., 2002) and multiplex-PCR (Yamasaki et al., 2004). 10

To be amenable for molecular identification using DNA detection, faecal samples must be fixed in ethanol or kept frozen (Yamasaki et al., 2004). The relatively low prevalence, 1% to 3%, at which taeniosis is often observed (Coral-Almeida et al., 2015), increases the requirements of diagnostic tools for T. solium taeniosis. Currently, there is a need for a highly specific and sensitive diagnostic test for taeniosis that does not cross-react with other parasites. It should be cheap, commercially available, and applicable within the field in rural areas of Africa, and therefore, not require advanced laboratory equipment. The assays described above are not readily available as they have not been commercialised. Usages are dependent on obtaining reagents from specific laboratories, and the majority of the assays require access to wellequipped laboratories, thus lowering the applicability in rural areas of Africa. Porcine cysticercosis Clinical signs of porcine cysticercosis caused by T. solium have not been widely researched, but can manifest as cysts on the tongue or in the eyes of pigs. Eye blinking, tearing, and excessive salivation have been suggestive of porcine cysticercosis when nodules are located in the pigs’ brain (Prasad et al., 2006). Inflammatory responses occurring around brain cysts could be decisive in the manifestation of symptoms (Mkupasi et al., 2013b; Sikasunge et al., 2008b; Singh et al., 2013). Trevisan et al. (2016) showed that pigs with porcine cysticercosis could develop clinical signs and suffer from seizures comparable to that seen in humans suffering from NCC. These seizures were seen in older pigs and later associated to the deposition of collagen around the cysts (Christensen et al., 2016). Pigs with brain cysts could prove to be an excellent model for neurocysticercosis (Trevisan et al., 2016). Clinical signs in pigs do however not occur at a frequency that is practical for diagnosis of porcine cysticercosis for screening purposes, due to the relative short lifespan of pigs when kept as livestock. Diagnosis of porcine cysticercosis can be done either ante-mortem or post-mortem. Lingual examination for cysts is highly specific, but the sensitivity is low and will also depend on the experience of the investigator (Phiri et al., 2002), and therefore underestimate disease occurrence. The likelihood of lingual cyst manifestation in pigs with porcine cysticercosis does increase with infection intensity (Lightowlers et al., 2015; Sciutto et al., 1998). A study showed that in naturally infected pigs up to 6% of cysts are located inside the tongue (Lightowlers et al., 2015), meaning less than 6% of the cysts would be detectable by lingual palpation. However, lingual examination can be used as a rapid tool for screening large numbers of pigs for ‘cysticercosis hot 11

spots’ (Guyatt and Fevre, 2016). This approach is probably best suited for highly endemic areas where high intensity infection is expected to occur more frequently compared to low endemic areas. Although sensitivity is low, lingual examination is practical, inexpensive, and can be performed anywhere, thus making the method highly applicable under field conditions. There are two monoclonal antibody-based ELISA assays available for detection of circulating antigens in pigs for research purposes; the B158/B60 (Brandt et al., 1992) and the HP10 (Harrison et al., 1989). The HP10 has been reported to have an approximate sensitivity and specificity of 55% and 83%, respectively (Krecek et al., 2008; Krecek et al., 2011), but in low intensity infections the sensitivity of the assay drops (Sciutto et al., 1998). The B158/B60 ELISA has been reported to have an approximate sensitivity and specificity of 87% and 95%, respectively, and to be able to detect as little as one viable cyst within infected pigs (Dorny et al., 2004). In experimental infected pigs with heavy infections, antigens have been detected with the B158/B60 as early as two weeks post infection, and at four to six weeks in pigs with light infections (Nguekam et al., 2003a). Using the B158/B60 a correlation between optical density (OD) values and infection intensity, as well as, fast antigen clearance after treatment, have been shown (Gonzalez et al., 2015). A recent study from Madagascar comparing the B158/B60 and the HP10 assays suggested that the B158/B60 assay is superior to the HP10 assay for epidemiological surveys on porcine cysticercosis (Porphyre et al., 2016), which is comparable to previous findings from South Africa (Krecek et al., 2008; Krecek et al., 2011). Drawbacks of both assays in terms of epidemiological surveys is that they cross-react with T. hydatigena (Devleesschauwer et al., 2013), and the production of monoclonal antibodies is limited, as availability is solely from institutional research laboratories. ApDia, Belgium, Reference 650501 has developed a commercialised kit for detection of cysticercosis in both pigs and humans based on B158/B60 assay, but the kit remains to be validated. Several methods of detecting antibodies specific for porcine cysticercosis have been described (Deckers and Dorny, 2010). Some, such as the ELISA’s have lower specificity (Tsang et al., 1989) and even suspected to cross-react in pigs with toxoplasmosis and trichinellosis (Ramahefarisoa et al., 2010), while others such as the enzyme-linked immuneelectrotransfer blot (EITB) assay (Tsang et al., 1991) are highly specific for T. solium cysticercosis. Common for all antibody assays is that they indistinguishably detect exposure, current or past infections, or even aborted infections (Garcia et al., 2001; Rodriguez-Hidalgo et al., 2006). None of the assays have been commercialised and they remain available only at specific laboratories for research purposes. 12

Molecular tools for porcine cysticercosis confirmation using PCR have been developed, but not standardised, for detecting DNA in cysts (González et al., 2006) and serum (Ramahefarisoa et al., 2010). Ramahefarisoa et al. (2010) showed that detecting DNA using nested PCR in sera from heavily T. solium infected pigs (5 cysts/250 grams of meat) had relatively low sensitivity (23-64%). PCR is currently not applicable as a diagnostic tool for screening, but remains a valid confirmatory tool of T. solium cysts. Post-mortem examination has good specificity, but the sensitivity will depend on infection intensity, number of incisions made, and the skill of the investigator (Sciutto et al., 1998). Carcass inspection as a diagnostic tool can have very low sensitivity if few cuts are made (Dorny et al., 2004). Legislation and guidelines for inspection of carcasses vary widely between regions, which complicates comparisons of post-mortem results between studies presenting carcass inspection data. One advantage with carcass inspection is that surveys can potentially be carried out without interfering with the transmission dynamics or the pork production chain if studies follow local carcass inspection standards at slaughter slabs. Full carcass slicing could be considered as the ‘gold standard’ for parasite detection and should theoretically have perfect sensitivity if the investigators are qualified, but it is also extremely tedious, time consuming, costly, requires a large team of trained personnel, and unsuitable in terms of meat inspection, as all meat is sliced and discarded. Lightowlers et al. (2015) suggested that partial carcass dissection consisting of slicing of the heart, tongue, and masticatory muscles are a reasonable sensitive method (approximately 80%) of detecting porcine cysticercosis compared to full carcass dissection. Partial carcass dissection lowers time and cost requirements substantially, and in terms of sensitivity comparable to immunodiagnostics. For all post-mortem examinations dubious cysts found will have to be confirmed by PCR (González et al., 2006) to gain excellent specificity. For assessing and monitoring interventions, post-mortem diagnostics has the drawback of requiring removal of pigs from the population. In doing so, the risk of inadvertently affecting transmission parameters dependent on population structures increases, thus, potentially cloaking the effects of the measured intervention. In addition, it is not always a given that pigs can be acquired for slaughter. Currently there is no consensus of a ‘gold standard’ diagnostic tool, neither for taeniosis or porcine cysticercosis. The development of a commercial point-of-care test highly specific and sensitive for T. solium cysticercosis in pigs is highly warranted. The aforementioned commercial test by ApDia, Belgium, 13

Reference 650501, is relatively expensive, requires relatively expensive laboratory equipment and a skilled technician, thus, decreasing the kits feasibility in the field. A cheap point-of-care test that could be used during meat inspection at slaughter slabs or even at the household level by unskilled personal could drastically improve the options for monitoring intervention strategies. Human cysticercosis In humans, cysts of T. solium can be found in the brain, spine, eyes, heart, skin and skeletal muscles (Garcia et al., 2003b; Rahalkar et al., 2000). Human cysticercosis is characterised by causing few symptoms, but can cause severe neurological disorders if the cysts are located in the central nervous system (NCC) (Garcia et al., 2003b), and has been reported to cause deaths (Mafojane et al., 2003). Clinical symptoms of NCC are nonspecific and vary according to cyst numbers and location, persistent and increasing headache is common. In severe cases, symptoms of hydrocephalus and increased intracranial pressure may occur with cysts in the ventricular system (Garcia et al., 2014). Strong associations between epileptic seizures and NCC have been reported (Montano et al., 2005; Mwanjali et al., 2013). Children and adolescents (age 3 to 24) with NCC are more likely to experience seizures when live or degenerating cysts are in parenchymal locations, and the risk of experiencing seizures increases with the number of cysts present (Kelvin et al., 2011). Neurocysticercosis is commonly asymptomatic the first five years after initial infection, but the period can vary from one year to more than 30 years (Kurrein and Vickers, 1977). Other symptoms of human cysticercosis are subcutaneous nodules, although more commonly manifested in people with cysticercosis from Asia and Africa (Garcia et al., 2003b). The consensus is that diagnostic criteria of cysticercosis require an evaluation of the combination of clinical presentation, neuroimaging, and serological tests (Del Brutto et al., 2001; Del Brutto, 2012; Del Brutto et al., 2017). Misdiagnoses have been reported of cysts thought to be T. solium, but in fact turned out to be other Taenia species (Taenia crassiceps and Taenia serialis) when molecular confirmation was obtained (Tappe et al., 2016). Diagnosis of NCC is done using imaging techniques such as computerized tomography (CT) or magnetic resonance imaging (MRI) scans and often done in combination with serology for antibodies or antigens (Deckers and Dorny, 2010). For the diagnosis of NCC, CT is preferable to detect calcified cysts, whereas, MRI is superior in detecting vesicular cysts (Verma and Lalla, 2012). As clinical manifestations for NCC are unspecific, a set of criteria for diagnoses based on both neuroimaging and serology (Brandt et al., 1992; Harrison et al., 1989; Tsang et al., 1989) on 14

serum or cerebrospinal fluid (CSF), has been suggested (Del Brutto et al., 2001; Del Brutto, 2012). But in principle, neuroimaging is essential for the diagnosis (Del Brutto et al., 2017). Neuroimaging requires specialised equipment and comes at high costs, and therefore, not available in many T. solium endemic areas. Serology can consist of either measuring antibodies using EITB or ELISA (Sako et al., 2013; Tsang et al., 1989; Tsang et al., 1991), or antigens using ELISA (Erhart et al., 2002; Garcia et al., 2000; Zea-Vera et al., 2013), and is not specific for neurocysticercosis, but for human cysticercosis. Subcutaneous cysticercosis can be diagnosed by physical examination for detection of nodules, and the diagnosis can be confirmed with biopsy or fine-needle cytology of the nodule (Sahai et al., 2002). Without confirmation misdiagnosis can occur and Katabarwa et al. (2008) reported that nodules thought to be onchocerciasis were in fact T. solium cysts. Cysts can lodge in the eye causing ophthalmic lesions leading to ocular cysticercosis which is the most common intraorbital parasite of humans (Rahalkar et al., 2000). Ocular cysticercosis can cause visual disturbance or even blindness, due to damage to retinal tissue, or the development of chronic uveitis (Cardenas et al., 1992), and is diagnosed using orbital ultrasonography (Rahalkar et al., 2000). Several commercial kits have been developed to detect cysticercosis antibodies in human serum for the diagnosis of cysticercosis. Five of these kits have shown relatively low sensitivity in one small study, but validation data was missing for all five kits (Carod et al., 2012). As current commercial kits detect antibodies, there is a risk of primarily estimating people that have been exposed instead of people with actual infections (Praet et al., 2010; Rodriguez-Hidalgo et al., 2006). Alternative approaches to diagnose human cysticercosis and more feasible sampling collection methods are sought. Urine has in addition to serum been investigated as a possible material for determining human cysticercosis by measuring antigens. However, the specificity of the assay is still low and requires more work before it is field applicable (Mwape et al., 2011). Filter paper has successfully been used to collect human blood samples from where antibodies for cysticercosis were detected (Jafri et al., 1998), but this sampling method is currently not widely used. Recently monoclonal antibodies against T. solium cyst antigens have been generated, and these show no cross-reaction with related parasites except for T. saginata in some cases, and were able in ELISA to detect infection in human blood and urine (Paredes et al., 2016). The assay needs to be validated and the sensitivity and specificity estimated. This new assay could potentially become important, also, for establishing infection in pigs as it does not seem to cross-react with T. hydatigena. 15

2.5 Intervention tools and control Current intervention tools with the aim of breaking the T. solium life cycle, targets either the human-to-pig or the pig-to-human transmission, with the exception of health education capable of targeting both simultaneously. Human-to-pig transmission can be intervened by providing preventive chemotherapy with anthelminthic drugs to humans, by improving pig production, by administration of pig vaccination, or by health education. Pig-to-human transmission can be targeted by anthelminthic drug treatment of pigs, by implementation of effective meat inspection, by health education, or by implementation of meat processing effective at killing T. solium cysts. Preventive chemotherapy of humans Humans with taeniosis can be targeted with a single dose of niclosamide (2 g for adults and 50 mg/kg for children) or a single dose of praziquantel (10 mg/kg) (Gherman, 1968; Pawlowski, 1991). Albendazole can also be used, but has to be administrated for three consecutive days to be efficacious (Chung et al., 1991). Preventive chemotherapy can be given as MDA in a defined area to either the whole population or targeted at a defined sub-population. Drugs of choice for MDA are praziquantel or niclosamide due to the safety profile of these drugs and the single dose administration. Single intervention approaches of preventive chemotherapy have been trialled in Latin America. In Ecuador, a drop in porcine cysticercosis prevalence from 11.4% to 2.6% was observed 12 months after community-based praziquantel MDA with a coverage of 76% (Cruz et al., 1989). Diaz-Camacho et al. (1991) reported a drop in taeniosis prevalence from 1% to 0% after praziquantel MDA with coverage of 71% in an endemic area in Mexico. However, prevalence was determined by coprology and the sample size was insufficient to allow for any conclusion to be made about the actual effect of the MDA. Also from Mexico, another study reported a decrease from 1.1% to 0.5% in taeniosis prevalence six months after praziquantel MDA with a coverage of 87% based on copro-Ag-ELISA. The prevalence of taeniosis remained at this level 42 months after the initial intervention without any additional interventions implemented, but was not significantly different from baseline (Sarti et al., 2000). A significant reduction in both taeniosis prevalence measured by microscopy, and porcine cysticercosis prevalence measured by EITB, was obtained by community-based niclosamide MDA with 75% coverage in Guatemala 10 months post intervention (Allan et al., 1997). There are currently no studies from sub-Saharan Africa assessing the effect of preventive chemotherapy administrated for taeniosis. The fact that praziquantel is effective against both 16

schistosomiasis and taeniosis, presents an opportunity for integrated control of these two helminth diseases. Praziquantel MDA is carried out in many areas of sub-Saharan Africa in efforts to control schistosomiasis and is expected to be further up-scaled in the near future (WHO, 2015a). So far no large-scale studies have evaluated the effect of integrated control of schistosomiasis and taeniosis. A study was recently carried out with the aim to assess the effect of integrating control of taeniosis with an existing national schistosomiasis control programme in two districts of Tanzania (Braae et al., 2016b; Braae et al., 2017). The study indicated that annual school-based praziquantel MDA for schistosomiasis was able to initially reduce taeniosis prevalence from 2.3% to 0.3% and maintain infection at a lower prevalence (0.1%) within the target population. In addition, the study suggested a significant reduction of taeniosis prevalence within the non-target population and the porcine population after three years of intervening if MDA was carried out annually (see section 3.3.4). The study supported the evidence from Latin America that preventive chemotherapy is able to reduce prevalence within endemic areas down to a certain threshold, but that transmission can still occur. Preventive chemotherapy is therefore on its own insufficient as an intervention tool to terminate transmission of the parasite, but as indicated by Braae et al. (2016b) a valid control tool. Alexander et al. (2011) estimated from a small study in India, based on 2008 prices and currency rates, that the cost of treating a taeniosis case using niclosamide MDA was 2,791 USD if the prevalence was 0.3% and 34 USD if the prevalence was 9.7%, as MDA becomes more cost-effective as prevalence increases. Improved pig production Improved pig production systems such as proper confinement whereby pigs are prevented direct access to human faeces should theoretically decrease the potential risk of infection to pigs. Sarti et al. (1997) reported that following a health education campaign fewer pigs were allowed to roam, which might explain the decrease in porcine cysticercosis measured by EITB, which was observed after the intervention. Data presented by Braae et al. (2015a) suggest that improving pig production systems such as maximising confinement might not have a high impact on prevalence of porcine cysticercosis in certain settings (see section 3.3.2). Focus should therefore also be put on pig management, such as securing clean and safe food and water, and proper living conditions. So far the effect of improved pig production has not been assessed.

17

Pig vaccination Several vaccines have been developed against T. solium cysticercosis in pigs (Sciutto et al., 2008), but currently only two vaccines appear to have the potential to control cysticercosis; the SP3Vac (Huerta et al., 2001) and the TSOL18, both based on a recombinant protein that is normally expressed in the oncosphere life cycle stage of T. solium (Gonzalez et al., 2005). Field studies from Mexico have shown that the SP3Vac significantly lowers the prevalence of porcine cysticercosis (Morales et al., 2008; Morales et al., 2011). The TSOL18 vaccine is predicted to be effective when given twice at a four month interval (Lightowlers, 2013), and yielded promising results under experimental conditions given in two doses to pigs approximately 12-weeks of age (Lightowlers et al., 2016). As pigs may become infected before they can be vaccinated (de Aluja and Villalobos, 1998), treatment with oxfendazole is necessary with the last injection. TSOL18 is currently being commercialised by Indian Immunologics Ltd with support from GALVmed, and will likely be the first commercially available vaccine against porcine cysticercosis caused by T. solium. A field trial was conducted in Cameroon to assess the TSOL18 vaccine, where pigs were vaccinated at the age of two to three months, inoculated with a booster shot combined with 30 mg/kg oxfendazole four weeks later, and another booster shot at 16 weeks post first vaccination (Assana et al., 2010). The study proved the vaccine to be 100% efficacious, but so far the effectiveness has not been evaluated. The obvious drawback of the TSOL18 vaccine are the requirements of a booster vaccination (Jayashi et al., 2012b), and the need to identify pigs with the primary vaccination eligible for a booster within four months. This might prove impractical in some rural areas and could lower the effectiveness substantially. Maternal antibodies against cysticercosis can be transferred to suckling piglets via the colostrum and start to decline four to six weeks after weaning, but can persist even 27 weeks after weaning in some cases (Gonzalez et al., 1999). Presence of maternal antibodies would exacerbate the problems of vaccinating younger pigs, in addition to their immune system not being fully developed (Lightowlers, 2010). This may reduce the efficacy of the vaccine and should be considered when deciding the age to give the vaccination series. Pig treatment Oxfendazole given orally at 30 mg/kg is currently the most efficacious anthelminthic for treating porcine cysticercosis (Mkupasi et al., 2013a). Treatment with oxfendazole has shown to reduce viable cysts by 50% one week after treatment and render all cysts located in the muscles non-viable 4 weeks post treatment, but had less than 100% efficacy against cysts located within the brain 18

(Sikasunge et al., 2008a). A 100% efficacy of oxfendazole against muscle cysts has also been shown to take more than 4 weeks, and only be fully efficacious at 12 weeks post treatment (Gonzalez et al., 1998). A randomised controlled field trial in Mozambique showed a significant drop in risk of porcine cysticercosis in 12 months old pigs treated with oxfendazole at either the age of four months or nine months, compared to untreated controls (Pondja et al., 2012). In infected pigs, cysts will have cleared from the muscle 6 months post treatment with oxfendazole (Sikasunge et al., 2008a). In addition to clearing cysts, oxfendazole is also efficacious against intestinal nematodes (Mkupasi et al., 2013a). Due to the withdrawal period of oxfendazole from tissues, pork from treated pigs is unfit for human consumption if slaughtered less than 17 days after the administration of oxfendazole (Moreno et al., 2012). Although these results are based on extrapolation of the data obtained only up to 96 hours post administration and therefore in need of validation. Infected pigs treated with oxfendazole were immune to reinfection of T. solium at least three months after treatment (Gonzalez et al., 2001), but the exact causality is unknown. Unfortunately, oxfendazole is currently not registered for use in pigs in sub-Saharan Africa, and therefore only remains available for research purposes. The effectiveness of oxfendazole as an intervention tool has yet to be evaluated. Meat inspection and processing Proper meat inspection with condemnation of infected pork should theoretically decrease transmission within an area with high infection intensities. Lack of compensation for condemned meat could be the biggest hurdle for setting up effective meat inspection strategies. Pigs suspected of infection are often slaughtered at clandestine slaughter slabs and pigs found infected at official slaughter slabs are often brought back to the farmers from where the pigs were purchased (Gonzalez et al., 2003). Yet, there has been no evaluation as to what degree this is occurring within an endemic area. The effect of improving meat inspection needs to be assessed in a sub-Saharan setting. Infected meat can be rendered safe by sufficient freezing, cooking, or processing i.e. salting (Flisser et al., 1986; Rodriguez-Canul et al., 2002), but this has also not been evaluated as a feasible intervention tool. Health education and sanitation Health education should comprehensively target human-to-pig and pig-to-human transmission simultaneously by focusing on lowering the risk of contaminating the environment with tapeworm eggs and by reducing the risk of consuming infected pork, respectively. Several studies evaluating 19

whether health education impacted knowledge uptake and practise change have been carried out (Alexander et al., 2012; Mwidunda et al., 2015), but few have actually assessed whether health education had an impact on prevalence or incidence of taeniosis, human cysticercosis, or porcine cysticercosis. In Zambia a preliminary post-intervention assessment in nine villages to evaluate Community-Led Total Sanitation (CLTS) showed no effect on the prevalence of porcine cysticercosis 8 months after the intervention (Bulaya et al., 2015). The study by Bulaya et al. (2015) is missing a control for comparison, making speculations on inference complicated if temporal fluctuations in porcine cysticercosis prevalence are suspected to occur (Braae et al., 2014) (see section 3.3.2). Health education with a focus on hygiene and food preparation in the context of the life cycle of T. solium may prove effective. Ngowi et al. (2008) demonstrated in a randomised controlled field trial a decrease in porcine cysticercosis prevalence and an increase in knowledge about the parasite following a health education campaign in Northern Tanzania. Ngowi et al. (2009) reported in an evaluation of the study that health education had resulted in a 43% drop in the incidence of porcine cysticercosis in sentinel pigs within the intervention group. Unfortunately, there was a large dropout of farmers during the study due to the incapability of farmers to keep the sentinel pigs. This might have led to a selection bias where only more economically resourceful households, presumably better equipped to learn from health education, participated in the study, leading to an overestimation of the effect of the intervention. A study from Mexico showed that health education could significantly decrease the prevalence of porcine cysticercosis measured by EITB one year after intervention (Sarti et al., 1997). Unfortunately, although a decrease in taeniosis was reported, the starting prevalence was too low given the sample size to register a significant effect in humans. In India a study showed increased knowledge relating to T. solium taeniosis/cysticercosis after health education (Alexander et al., 2012), but no effect on prevalence of disease was investigated. A computer-based education tool for T. solium taeniosis/cysticercosis called “The Vicious Worm” was developed to be used as a specific evidence-based education tool in control programmes targeting T. solium (Johansen et al., 2014). The Vicious Worm was assessed in Tanzania and showed to increase the participants’ knowledge of T. solium significantly (Ertel et al., 2017). Standardising health education and recognising it as a specific tool in line with e.g. preventive treatment, would improve the ability of a more rigorous assessment of the impact of health education. Also important, health education programmes should include information on hygiene. Strict hygiene can prevent human 20

cysticercosis within a T. solium endemic area where tapeworm carriers are present, and hygiene might also be important in terms of perpetuating transmission of T. solium to pigs (Braae et al., 2015a) (see section 3.3.2). Adequate levels of sanitation will have an effect on a range of faecal borne diseases - taeniosis included - but seems far from being implementable in rural areas of low-income countries. Studies on improving water and sanitation have reported good effects on a number of other faecal borne parasites (Esrey et al., 1991). There has been advocacy for water, sanitation, and hygiene (WASH) to be an integrated component of NTD control (Freeman et al., 2013), but so far no studies have evaluated WASH in terms of T. solium, and the paucity of experimental evidence concerning this needs to be addressed. More studies assessing the cost-benefit and the efficacy of health education on the reduction of T. solium transmission are needed (Lightowlers, 2013). Combined intervention tools Two studies from Mexico combined MDA and health education against T. solium (Keilbach et al., 1989; Sarti et al., 1998). Keilbach et al. (1989) reported an increase in prevalence of porcine cysticercosis based on lingual examination from 7% to 11% one year after praziquantel MDA with 60% coverage combined with continuous health education activities within the community during the study period. The study was lacking a comparison group making it difficult to evaluate the impact of the intervention. In contrast, Sarti et al. (1998) reported that combining praziquantel MDA with health education successfully reduced prevalence of both taeniosis and porcine cysticercosis three years after implementation. However, details describing this were lacking making evaluation of the study difficult at best. In Peru, a randomised control study was carried out from 1996 to 1998 with the aim of assessing the effect of a combined intervention strategy targeting both humans and pigs. The strategy consisted of community-based praziquantel MDA with an estimated coverage of 75% and two rounds of oxfendazole MDA to pigs with an estimated coverage of 90% (Garcia et al., 2006). The authors concluded that the observed decrease in prevalence and incidence, based on EITB, of porcine cysticercosis was a result of the intervention and lasted up to 18 months post intervention. Medina et al. (2011) performed a comprehensive intervention study during an 8 year period in 18 Honduran villages using a multi-intervention approach with human treatment, health education, and improvement of sanitation. The intervention was associated with a decrease in the proportion of NCC associated epilepsy cases from 37% to 14% and the incidence of epilepsy being halved. The 21

study was limited by being non-randomised and based on baseline prevalence data that included all cases of active epilepsy, while follow-ups only included new onset seizure cases, leading to an overestimating of intervention effects. The proportion of taeniosis cases decreased from 2.8% to 0.3% based on Kato-Katz, but it is unclear whether this was significant. Garcia et al. (2016) unequivocally carried out the largest study to date in Peru from 2004 to 2008, presumably, as there was no mention of when the study ended. The study comprised of three intervention phases each 12 months in duration, and each with a different scale. The first phase included 42 villages and 10,753 humans and 17,102 pigs, the second phase 17 villages and 10,380 humans and 13,488 pigs, and the third and last phase included all 107 villages within the region and 81,170 humans and 55,638 pigs. The human and pig population denominators were unclear for each intervention, and the coverage of each intervention was therefore uncertain. The intervention tools applied within different villages and at different frequencies consisted of human MDA, screening of humans and pigs, monthly strategic treatment of pigs, health education, pig vaccination, and pig replacement, where infected pigs were bought and replaced with uninfected piglets. The effect of the three phases was evaluated by antibody detection and necropsy to determine presence of viable cysts in pigs six to eight months of age. At baseline the sero-prevalence based on antibody detection ranged between 25% and 50% at village level. Immediately after the first phase prevalence of porcine cysticercosis was recorded to 5.5% (18/326) based on necropsy, and 29% based on antibody detection. Immediately after the second phase the prevalence was 0.9% (6/658), 12 months after phase two and prior to further interventions the prevalence was 2.3% (7/310), and immediately after the third and last phase the prevalence was 0.9% (3/342). Nonetheless, the authors concluded to have interrupted transmission on a regional scale. There is no doubt that the investigated intervention strategies were effective in bringing down disease prevalence, but whether such an intensive programme would be cost-effective remains to be evaluated. Also, evaluation of how long effects persist after intervention has been terminated is also important, as this is currently not known, yet modelled simulations suggest that effects might be long lived (Braae et al., 2016a). A study from Laos targeting both hosts, combined a repeated 3-day albendazole MDA to the human population followed by oxfendazole treatment and vaccination of pigs, and reported a reduction in copro-Ag-prevalence of taeniosis from 23% to 6% one year after the initial intervention (Okello et al., 2016). As the aforementioned study from Peru (Garcia et al., 2016), the effect of the intervention was only measured within one host population – the human host. 22

Control of Taenia solium Guidelines for the control and surveillance of T. solium were first published more than 30 years ago (Gemmell, 1983), but T. solium remains a challenge in many low-income countries where extensive pig production systems occur. Control of an infectious disease has been defined as: “a reduction in disease incidence, prevalence, morbidity, or mortality to a locally acceptable level, as a result of deliberate efforts; continued intervention measures are required to maintain the reduction” (Dowdle, 1998). But for zoonotic agents, such as T. solium, control of more than one disease can be required e.g. both taeniosis and porcine cysticercosis. Acceptable levels of e.g. disease prevalence or incidence have not been put forward by any sub-Saharan country government. Local governments need data on the parasites’ geographical distribution, degree of endemicity, and burden estimates in order to justify policies targeting control of T. solium. Pushing through these policies requires One Health cross-sectoral collaborations recognising the problem, not only for human, animal, and environmental health, but also, for the economic burdens preventing some of the poorest people from moving out of poverty. Using prevalence or incidence of human cysticercosis as the dependent variable for estimating intervention effects might be both expensive and complicated by the potential delay in measureable effects from intervening with the intermediate or definitive host to the actual manifestation in human cysticercosis prevalence or incidence. Prevalence of porcine cysticercosis therefore remains the best option, followed by prevalence or incidence of taeniosis for monitoring the outcome of intervention measures within a T. solium endemic area. Intervention studies have been carried out aiming to reduce disease prevalence or incidence, but for control, local accepted levels of prevalence or incidence, and a timeframe for reaching and sustaining these levels, should be defined before control can truly be claimed to have been achieved. Despite the zoonotic properties of T. solium, a one host control strategy can theoretically be effective, but is less robust, requires excellent effectiveness, and a longer running phase to reach control goals compared to a One Health control strategy aiming to reduce prevalence in both parasite reservoirs simultaneously (Braae et al., 2016a) (See section 3.3.5). Obtaining transmission control with the existing intervention tools might be possible, and the next logical step (Gabriel et al., 2017), but complicated by the limits to what can realistically be measured in terms of prevalence or incidence with the existing diagnostic tools. Hence, moving from control to elimination will probably require field applicable, specific, sensitive, and cheaper diagnostic tools compared to those currently available.

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2.6 Transmission modelling to assess interventions for the control of Taenia solium Intervention studies are time consuming and costly. Transmission models can therefore be an alternative to investigate the effects of different intervention strategies. The need for a T. solium transmission model was emphasised already in 1989 (Gemmell and Lawson, 1989), but it was not until 2007 that the first model became available. The mathematical compartmental model by Kyvsgaard et al. (2007) was created to simulate the transmission of T. solium. It was developed both in a deterministic and stochastic version, and provided insight into the transmission dynamics of T. solium. The model predicts significant impacts of the most common intervention strategies, but also returns towards a steady state once interventions are terminated. When modelling intervention strategies in this model, users have to specify the reduction in transmission probability for each specific transmission point e.g. pig-to-man transmission which is not always straightforward. On the other hand, this makes the model extremely flexible and creates the option for modelling any type of intervention as long as the user predicts the reduction in the transmission probability caused by the intervention e.g. improved sanitation can reduce the man-to-pig transmission probability by 50%. The main drawbacks of the model are the lack of age structures, environmental aspect, spatial dimension, and the fact that it is parameterised based on data that originated from different endemic areas, adding variation to the model. In advocating for T. solium control the model is useful to illustrate which intervention approaches might have impact on disease prevalence of taeniosis and porcine cysticercosis, but the model lacks the ability to estimate cases of human cysticercosis adverted, which could further move the model from being a research tool to an advocacy tool. cystiSim, a recently developed agent-based model for T. solium transmission and control simulation, includes age structures, parasite maturation, host immunity, and environmental contamination (Braae et al., 2016a). The model builds on data from Tanzania, but can accommodate any endemic setting by changing the underlying baseline data. Advantages, limitations, and examples of intervention simulations are discussed in section 3.3.5.

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2.7 Justification The population growth in Africa is currently estimated to be higher than in any other continent, and Africa is the only continent, based on projections, that will still see large population increases even in the year 2100 (Gerland et al., 2014). These population projections for Africa estimate that the approximately one billion people of Africa today, will increase to more than two billion in the year 2050. This will cause the demand for pork in Africa to drastically increase within the next decades (Delgado, 2003). In meeting this demand it is of vital importance that focus is put on improving the agriculture and livestock sectors within Africa, so these sectors are able to provide sufficient amounts of safe food, e.g. pork, for the consumers. Taenia solium currently poses a threat to both food security and food safety in sub-Saharan Africa and a current problem (Braae et al., 2015d) (see section 3.3.1). With the projected increases in human populations and the necessary increases in porcine populations to meet demands, the potential for emergence of T. solium in new areas is conjecturally great. In Tanzania, school-based praziquantel MDA is carried out as part of the National Schistosomiasis Control Programme (NSCP) in schistosomiasis endemic districts such as e.g. Mbozi and Mbeya in the Southern Highlands. The impact and cost-effectiveness of integrating schistosomiasis control with control of T. solium is unknown, and hence, the need is great for monitoring on-going pilot integrated intervention programmes in Africa addressing this. The integrated approach of MDA against several NTD is now recognised as a safe and important tool in improving global public health (Hotez, 2009), but the safety of praziquantel MDA in T. solium endemic areas needs to be further assessed. The variation in sensitivity and specificity of existing diagnostic tools for taeniosis, human and porcine cysticercosis, and the lack of uniform protocols for assessing infection prevalence and incidence, make comparisons between different studies on T. solium, or even measuring effects of a control programme, difficult. So far comparable information on porcine cysticercosis prevalence over time obtained using the same methodology from the same area in sub-Saharan Africa is scant. Therefore, estimates on whether the level of porcine cysticercosis endemicity is stable over time or if seasonal variations in prevalence exist needs further attention. Potential temporal fluctuations in prevalence may have consequences for the timing of intervention implementation against T. solium as well as the comparison of prevalence data. In order to help achieve transmission control of T. solium taeniosis/cysticercosis an algorithm consisting of the right combination of feasible 25

intervention tools needs to be created. Models simulating the transmission of the parasite, could aid in identifying the best bet option for the combination of such intervention tools. This could guide the design of large-scale control programmes, likely saving time and vast amounts of resources before they are implemented.

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3. Own investigations 3.1 Objectives The overall objective was to describe the epidemiology and the potential for control of T. solium, by investigating parasite transmission dynamics, assessing the potential for integrated control within existing programmes such as schistosomiasis control, and creating a transmission model to evaluate potential intervention tools, thereby contributing towards future design and implementation of feasible and sustainable control efforts against T. solium in low-income countries. Specific objectives: 1) To map the district level distribution of T. solium and the co-distribution of T. solium with schistosomiasis on a national scale in Africa 2) To investigate possible temporal fluctuations in porcine cysticercosis prevalence and identify risk factors for porcine cysticercosis among smallholder farms with persistent or multiple infections in Mbeya and Mbozi district, Tanzania 3) To determine the prevalence of T. hydatigena among slaughtered pigs in Mbeya, Tanzania 4) To assess the effect of repeated school-based praziquantel MDA in combination with ‘trackand-treat’ on taeniosis and porcine cysticercosis in a T. solium endemic area of Tanzania 5) To create an agent-based model based on data from sub-Saharan Africa capable of evaluating potential intervention strategies against T. solium

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3.2 Materials and methods - limitations Taenia solium presence has been reported in Tanzania from several districts, mostly based on porcine cysticercosis prevalence (Boa et al., 1995; Boa et al., 2002; Boa et al., 2006; Komba et al., 2013; Ngowi et al., 2004a; Ngowi et al., 2004b; Ngowi et al., 2010), but also based on taeniosis and NCC cases (Blocher et al., 2011; Hunter et al., 2012; Hunter et al., 2015; Mwanjali et al., 2013). The two contiguous districts Mbeya and Mbozi were found to be endemic for T. solium taeniosis/cysticercosis from 2008 (Kabululu et al., 2015; Komba et al., 2013; Mwanjali et al., 2013), and therefore chosen as the study area. The human population was by census estimated in 2012 to be 305,319 in Mbeya district and 446,339 in Mbozi district (URT, 2013). Pig production within the two districts is almost exclusively on a smallholder level, and was in 2007/2008 estimated to 31,190 pigs in Mbeya district and 117,483 pigs in Mbozi district (URT, 2012). The work presented in this thesis was carried out in 11 villages in Mbozi district, 13 villages in Mbeya district, and at a slaughter slab (Mbalizi) located in Mbeya district (Figure 3).

Figure 3: Study area, Mbeya and Mbozi district in Tanzania, showing where human stool and porcine blood samples were collected in the period 2012 to 2015 and where pigs where examined during the slaughter slab survey.

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In order to estimate taeniosis prevalence, 1,500 human participants willing to provide a stool sample within the study villages in each district were targeted during each survey, with the following age distribution: 4-15 (30%), 16-25 (30%), 26-40 (30%), and >40 (10%). If an insufficient number of participants were located within a specific age group, the study was limited by including more participants from other age groups in order to reach the district target of at least 1,500 people. Laboratory examinations Stool samples were collected and placed in 10% formalin for taeniosis prevalence estimation. Taenia solium antigens stay stable for several months if preserved in formalin 5-10% at room temperature (Allan and Craig, 2006). Stool samples were analysed for taeniosis infections using copro-Ag-ELISA (Allan et al., 1990). The copro-Ag-ELISA is incapable of distinguishing between infections of T. solium and T. saginata. In our studies no attempts were made to differentiate whether positive cases by copro-Ag-ELISA where in fact T. solium or T. saginata infections. However, T. saginata has not been reported in the area and beef was not seen to be consumed within any of the study villages. Porcine serum samples were analysed using the B158/B60 Ag-ELISA (Brandt et al., 1992; Dorny et al., 2004; Sikasunge et al., 2007) to estimate the prevalence of porcine cysticercosis. Each serum sample was analysed in duplicate, and the average OD value divided by the cut-off value to yield the ratio – ratio values larger than 1 constituted a positive sample. The cut-off value was based on eight negative reference samples using a variation of the Student’s t-test (p=0.001) (Sokal and Rohlf, 1981). The B158/B60 Ag-ELISA assay results presented in this thesis were limited by the use of negative reference samples from a population different from the population under investigation. Sensitivity and specificity of a given diagnostic test are independent of the population subjected to the test, but in terms of ELISA assays, the cut-off value of the OD values are dependent on the reference population (Greiner and Gardner, 2000). Due to the lack of an ante-mortem gold standard test with 100% sensitivity and 100% specificity, it is not possible to obtain negative reference samples from a T. solium endemic area in Tanzania. Also, the assay has high repeatability when evaluated based on positive (T. saginata) bovine serum samples (Jansen et al., 2016), but is incapable of distinguishing between T. solium and T. hydatigena infections in porcine samples (Devleesschauwer et al., 2013; Dorny et al., 2004). No distinctions were made to establish whether positive pigs were infected with T. solium or T. hydatigena in the field surveys. We assumed that the infection pressure of T. hydatigena was identical between the two districts. 29

3.3 Results and discussion 3.3.1

Distribution of Taenia solium and the co-distribution with schistosomiasis in Africa (Paper I)

Taenia solium is still presumed to be grossly underreported in sub-Saharan Africa. It has from 1985 to 2014, been reported in 31 countries based on published literature and OIE reports (Braae et al., 2015d). This is the first published district level distribution map of T. solium in Africa, with reports from 476 districts across the continent (Figure 4). Porcine cysticercosis was reported in 20 countries of which seven countries had no reports of taeniosis, which highly warrants epidemiological surveys to investigate taeniosis prevalence in those seven countries. In addition, no data on T. solium were found for 10 African countries known to keep pigs. Taeniosis was reported in 16 countries and human cysticercosis in 22 countries although only autochthonous cases were included. Active transmission of the parasite between humans and pigs was unconfirmed in the five countries where only reports of human cysticercosis were found and no reports of taeniosis or porcine cysticercosis were available. The discrepancy between the number of countries with taeniosis and human cysticercosis illustrates that more information on T. solium is still urgently needed.

Figure 4: Distribution of Taenia solium on district level in Africa (Braae et al., 2015d).

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Taenia solium taeniosis/cysticercosis and schistosomiasis was considered co-distributed on a national level in all countries where T. solium was reported (Figure 5). Combining data from the Global Neglected Tropical Disease Database (GNTD; www.gntd.org) with the T. solium taeniosis/cysticercosis district map (Figure 4) revealed the presence of both diseases in 124 districts (second-level administrative division) in 17 countries. The co-distribution of T. solium taeniosis/cysticercosis and schistosomiasis in Africa emphasises the importance, and the need, to evaluate integrated intervention approaches targeting both helminth infections. National schistosomiasis control programmes using 40 mg/kg praziquantel as preventive treatment could potentially be more cost-beneficial than currently estimated if evaluation of the benefit was done using a One Health approach to include all diseases susceptible to praziquantel, instead of just schistosomiasis. In Tanzania, the NSCP has intensified distribution of praziquantel to school-aged children with the inclusion of the districts Mbeya and Mbozi in August 2012. The two districts fall under two different treatment regimens with annual school-based praziquantel MDA in Mbozi district and biennially school-based praziquantel MDA in Mbeya district.

Figure 5: Co-distribution of Taenia solium infections and schistosomiasis on a national level in Africa based on published literature from 1985 to 2014 (Braae et al., 2015d).

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The results presented by Braae et al. (2015d) highlight, that basic epidemiological surveys investigating parasite distribution, is still a valid research activity in most of sub-Saharan Africa and is highly warranted. For the majority of African countries where transmission of T. solium occurs, the distribution is either unknown or only known on a large scale such as on a regional scale. In order to estimate disease burdens, argue for national policy change, and implement control strategies in optimal areas, knowledge on parasite distribution is required. 3.3.2

Temporal fluctuations and risk factors of porcine cysticercosis (Paper II & III)

The presence of porcine cysticercosis in Mbozi and Mbeya districts in Tanzania has recently been confirmed (Komba et al., 2013). In order to control T. solium, an understanding of the transmission dynamics and the underlying risk factors is required. This can have implications for the timing of interventions and when the most beneficial effect is achievable. Therefore, from March/April 2012 to July/August 2013, based on the B158/B60 Ag-ELISA (Brandt et al., 1992; Dorny et al., 2004), the existence of temporal fluctuations was investigated in the two districts Mbeya and Mbozi (Braae et al., 2014). Data were collected at three sampling points during different agricultural seasons, suspected of affecting prevalence due to differences in the production systems primarily practised during each agricultural season. The sero-prevalence of porcine cysticercosis fluctuated throughout the study period. There were no differences however in proportions of pigs being confined in any of the surveys, but changes in proportions of semi-confined and free-range pigs. Contradictive to previous data from the region (Komba et al., 2013), confined pigs were just as likely to be infected with porcine cysticercosis as free-range pigs. Lack of association between production systems and porcine cysticercosis indicates one of three things: 1) within pen transmission occurred under the settings present at the time, potentially through contaminated feed and water, or 2) pigs were not truly confined, or 3) a combination of the two. Porcine cysticercosis sero-prevalence, although fluctuating, was still overall lower than previously recorded in the area (Komba et al., 2013). The higher sero-prevalence observed by Komba et al. (2013) could be explained by a larger proportion of free-range pigs at that time, also supporting their finding of association between the free-range production system and infection with T. solium in pigs. The increase in proportion of confined pigs compared to Komba et al. (2013), and perhaps the overall lower prevalence of porcine cysticercosis infection in pigs seen in our study, could be explained by farmers’ apparent reluctance to let pigs roam due to fear of African swine fever (ASF). The large outbreak of ASF in the area in 2010 prior to our study (Braae et al., 2015b), could have 32

killed a substantially larger proportion of free-range pigs compared to confined pigs, and in that process decreased the prevalence of porcine cysticercosis within the area. Unfortunately, Komba et al. (2013) did not provide information on pig production systems at district level, but did find that the prevalence of porcine cysticercosis in Mbozi district was approximately twice that of Mbeya district when measured by lingual examination. Perhaps, an indication of pigs’ direct exposure to faecal matter (free-range) versus indirect exposure to faecal matter (confinement) if we assume more pigs were free-range in Mbozi district compared to Mbeya district at the time, which was the case during our study (Braae et al., 2016b). Both studies (Braae et al., 2014; Komba et al., 2013) were limited by the lack of documentation on presence or absence of T. hydatigena within the study area, and the lack of knowledge about the transmission dynamics and potential temporal variation in prevalence of T. hydatigena. The pig population and the mean age of pigs increased during the study period, presumably due to restocking after the ASF outbreak in 2010. Increasing age was associated with T. solium infection during the last survey, indicating that pigs were unlikely to recover from infection, supportive of the findings by Nguekam et al. (2003a), perhaps because of pigs’ relative short life span as production animals. Experimental infections have shown that pigs’ susceptibility to infection with T. solium might decrease with age as fewer cysts were seen in older animals as per infection dose (de Aluja et al., 1996; Deckers et al., 2008). The finding that production systems played an insignificant role in the transmission of T. solium (Braae et al., 2014), led us to believe that transmission could potentially occur within the pig pens. We hypothesised that such transmission could be perpetuated by feed grown in a Taenia egg contaminated environment. Utilising data from Braae et al. (2014), smallholder farms with no record of porcine cysticercosis on at least two occasions (controls) and farms with persistent/multiple infections (cases) were identified. The case-control study consisted of a questionnaire and an observational survey (Braae et al., 2015a). The study revealed an association between cysticercosis sero-prevalence in pigs and missing or poor latrines, corresponding to findings of previous studies (Boa et al., 2006; Jayashi et al., 2012a; Ngowi et al., 2004a; Sarti et al., 1992a). Feeding pigs potato peels was also found to be a risk factor for porcine cysticercosis (Figure 6). Potato by-products have previously been associated with T. saginata infections in cattle in USA (Yoder et al., 1994). However, if potato peels were in fact contaminated with Taenia eggs, it is still speculative whether the contamination occurred from the environment on the field, or when 33

Figure 6: (A) People peeling potatoes in Idimi village, Mbeya district, and (B) potatoes peels fed to a pig in Jojo village Mbeya district

handled for storage or peeling by a tapeworm carrier. Well described epidemiological studies identifying and quantifying environmental contamination with T. solium eggs are still missing. What is needed in order to quantify the contamination is a description of how many viable eggs are present within a specific area as a function of how many tapeworm carriers are present over time. The studies that have been carried out so far investigating soil samples for presence of Taenia eggs have been unquantifiable or non-reproducible due to limited methodological descriptions (EdiaAsuke et al., 2014; Gweba et al., 2010). Previous field investigations of Taenia eggs in the environment have all been cross-sectional, and the potential for temporal fluctuation in quantities of eggs therefore unknown. Our study suggests that farm management might be an important factor in the transmission of porcine cysticercosis under the study settings. The choice of feed provided for the pigs might have an influence on disease status (Braae et al., 2015a). An association of different pen types and infection in pigs was also seen, similar to what was reported from the same area by Komba et al. (2013). The results of both studies underline that there is still something overlooked in terms of fully understanding the transmission to pigs, and that pig management might play a significant role in the transmission of porcine cysticercosis with potential consequences for the success of future intervention strategies and control programmes. Farmers within the area generally lacked knowledge on important issues of pig management (Braae et al., 2016c). Studies on feasible and realistic improvements of pig management in rural settings, and elucidating what impact this has on infection is needed.

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Areas where confinement is predominant and transmission perpetuated through indirect transmission such as through contaminated feed and water might have substantially lower infection intensities compared to areas where pigs are infected through direct transmission such as direct access to human faeces, which would lead to higher infection intensities. Similarities are known from T. saginata infections in cattle in Europe where heavy infections are quite rare unless the animals have been in direct contact with raw sewage (Dorny and Praet, 2007). The two scenarios, indirect and direct transmission settings, might differ in the requirements of intervention strategies and methods used to measure the impact of such strategies. For example, if many pigs have heavy infection burdens, then effective carcass inspection might be a valid tool, but if light infections are predominant carcass inspection will not suffice. Lingual examination could be a valid tool for surveillance in high risk areas (prevalence of above 30%) (Guyatt and Fevre, 2016). The relationship between true prevalence and the apparent prevalence found by lingual examination is highly dependent on type of production system present if this reflects the transmission setting e.g. indirect or direct transmission. Lingual examination as a surveillance tool will not perform well in areas where indirect transmission prevails, as it will underestimate prevalence to a larger degree than under direct transmission settings where more high intensity cases are found. Our studies show that potential fluctuations in porcine cysticercosis prevalence could be considered when designing studies or monitoring programmes where porcine cysticercosis prevalence is a dependent variable. Pig management should be considered as an important factor in intervention strategies, also in areas where indirect transmission occurs. 3.3.3

Presence of Taenia hydatigena - implications for diagnosis of porcine cysticercosis (Paper IV)

A slaughter slab survey was carried out in Mbeya district in April to May 2014 to investigate the presence of T. hydatigena (Figure 7). The study confirmed, with relatively high prevalence (6.6%), the presence of T. hydatigena in pigs (Braae et al., 2015c). Taenia hydatigena has previously been reported in pigs in Africa with similar levels of prevalence e.g. 6.1% in Zambia (Dorny et al., 2004), 6.7% in Ghana (Permin et al., 1999), and 8.8% in Burkina Faso (Dermauw et al., 2016). Only one study on T. hydatigena in pigs from Tanzania has previously been published were a prevalence of 1.4% was found (Ngowi et al., 2004b), which was comparable to an older Nigerian study that reported a prevalence of 2.2% in pigs (Fabiyi, 1979). However, these African figures are substantially lower than reports from Asia (Nguyen et al., 2016). The economic and welfare burden 35

Figure 7: (A) Pig carcasses being inspected at the Mbalizi slaughter slab in Mbeya district and (B) Taenia hydatigena cyst on the liver of a pig.

of T. hydatigena infections in pigs are unknown. In the context of diagnosing T. solium in pigs when using the B158/B60 Ag-ELISA (Brandt et al., 1992), T. hydatigena becomes important as the assay cross-reacts with T. hydatigena (Devleesschauwer et al., 2013; Dorny et al., 2003). Our study confirmed the presence of both T. hydatigena and T. solium in Mbeya, Tanzania, although, the prevalence of T. solium (1.2%) was low (Braae et al., 2015c). This low prevalence compared to previous studies (Braae et al., 2014; Komba et al., 2013) could be a result of pigs being prescreened for cysts by lingual examination before being sent for slaughter, thereby reducing the number of pigs slaughtered with high intensity infections. Prevalence of T. solium, even when factoring in T. hydatigena and the pre-screening of T. solium, was lower compared to previous accounts from surrounding villages (Braae et al., 2014; Komba et al., 2013). The study was limited by being executed within a slaughter slab and the results will likely be significantly different at a village level when taking all age groups of pigs into account. As pigs were examined during meat inspection, most low intensity infections with T. solium were likely missed (Dorny et al., 2004). With the presence of T. hydatigena in Mbeya Region, intervention efforts against T. solium, when evaluated on the basis of porcine cysticercosis measured by Ag-ELISA, could be underestimated. The consequence of this would be a necessary increase in sample size requirements if intervention efforts omit T. hydatigena. 3.3.4

School-based praziquantel MDA – effect on taeniosis and porcine cysticercosis (Paper V & VI)

Braae et al. (2016b) suggest that school-based praziquantel MDA targeting schistosomiasis can have an effect on both taeniosis and porcine cysticercosis if treatment is annual for at least three years. However, short-term the study revealed only an effect within the target population. In total, 36

the study comprised of 12,082 stool samples examined for taeniosis (Table 1) and 4,579 porcine serum samples examined for porcine cysticercosis (Table 2). Long-term, the study showed that there was a significant difference between the effect on porcine cysticercosis of annual MDA in Mbozi district and biennial MDA in Mbeya district (Braae et al., 2016b; Braae et al., 2017). The study showed that there is a potential added value of large control programmes utilising treatment which is effective against multiple pathogens. Table 1: Study population characteristics and prevalence of taeniosis in the two districts Mbozi and Mbeya at Feb-Apr 2012 (S0), Jul-Aug 2013 (S12), Jul-Aug 2014 (S24), and Jul-Aug 2015 (S36) Survey District

S0

S12

S24

S36

Mbozi

Mbeya

Mbozi

Mbeya

Mbozi

Mbeya

Mbozi

Mbeya

1519a

1510b

1500c

1521d

1514

1500

1517e

1501f

Males %

50

50

52

48

46

45

46

43

Females %

51

49

48

52

54

55

54

57

951

880

712

1098

1255

1400

1070

1076

Stool samples

Children (≤15) Adults (>15)

561

621

786

422

259

100

444

425

Taeniosis (%)

45 (3.0)

22 (1.5)

30 (2.0)

5 (0.3)

12 (0.8)

8 (0.5)

9 (0.6)

7 (0.5)

Taeniosis in children (%)

22 (2.3)

11 (1.3)

2 (0.3)

2 (0.2)

7 (0.6)

8 (0.6)

1 (0.1)

4 (0.4)

Taeniosis in adults (%)

23 (4.1)

11 (1.8)

28 (3.6)

3 (0.7)

5 (1.9)

0 (0.0)

8 (1.8)

3 (0.7)

Sex was not recorded for 7 individuals, Sex was not recorded for 9 individuals, Sex was not recorded for 2 individuals, Sex was not recorded for 1 individual, e Age was not recorded for 3 individuals, f Sex was not recorded for 1 individual a

b

c

d

The potential added value of integrating control of other diseases (Hotez, 2009) when deciding which areas to include in control programme activities, or when estimating the cost-effectiveness or cost-benefit of a programme, needs attention. Cost-benefit analyses are mostly suitable for comparing within country programmes performed under the same overall conditions, whereas, comparison of national control programmes should be done using cost-effectiveness analyses. Treatment of humans has been carried out in Latin America using a single MDA of either praziquantel (10 or 5 mg/kg) (Cruz et al., 1989; Diaz-Camacho et al., 1991; Sarti et al., 2000) or niclosamide (2 g ≥ 6 years, 1 g < 6 years) (Allan et al., 1997) as the intervention, with the outcome measured on both human and porcine variables. Allan et al. (1997) carried out ‘track-and-treat’ in two communities followed by MDA which revealed a drop in both taeniosis and porcine cysticercosis based on copro-Ag-ELISA and EITB, respectively. However, the study lacked comparison between groups and options for assessing different treatment strategies. Sarti et al. (2000) showed from EITB results, a reduction in porcine cysticercosis prevalence 6 months after 37

treatment, but saw no significant change after 42 months. Taeniosis prevalence based on copro-AgELISA and egg detection was lower after 6 months and 42 months, but not significantly different from baseline. The study lacked a control group or comparison between interventions. Both Cruz et al. (1989) and Diaz-Camacho et al. (1991) reported a decrease in taeniosis and porcine cysticercosis prevalence, but unfortunately the studies were either based on a small sample size or lacked a control group. A large study in Northern Peru showed significant reductions in prevalence of porcine cysticercosis following implementation of a multiple intervention strategy (Garcia et al., 2016). However, the strategy was unable to completely interrupt transmission to pigs, stated as the overall goal of the study. Children from Mbozi who had received MDA annually showed significantly decreased odds of being infected throughout the study period (Figure 8). In Mbeya, the children that had received biennial MDA only had significantly decreased odds of infection after the first MDA. Among the children in Mbeya decreasing age was associated with infection. This could be explained by younger children being present for fewer treatments as they would not have started school yet during the first MDA rounds and therefore more likely to have missed treatments.

Figure 8: Taeniosis prevalence among children (4-15) and adults (≥16) in areas receiving school based MDA in Mbeya and Mbozi district, Tanzania at Feb-Apr 2012 (S0), Jul-Aug 2013 (S12), Jul-Aug 2014 (S24), and Jul-Aug 2015 (S36). Asterisk (*) marks significant difference with baseline (S0) based on logistical regression with *p=0.01-0.05, **p=0.001-0.009, and ***p