control of taenia solium in zambia · taenia solium en behoort tot de ‘verwaarloosde tropische...

CONTROL OF TAENIA SOLIUM IN ZAMBIA Word count: 33439

Tine De Coster Student number: 01203585

Supervisor: Prof. dr. Sarah Gabriël Supervisor: Dr. Kabemba E. Mwape A dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of Master of Veterinary Medicine Academic year: 2017 - 2018

Ghent University, its employees and/or students, give no warranty that the information provided in this thesis is accurate or exhaustive, nor that the content of this thesis will not constitute or result in any infringement of third-party rights. Ghent University, its employees and/or students do not accept any liability or responsibility for any use which may be made of the content or information given in the thesis, nor for any reliance which may be placed on any advice or information provided in this thesis.

Preface The composition of a master thesis is the final chapter in obtaining the degree of veterinary medicine. When choosing to graduate in research, this master thesis becomes of major importance. I had the opportunity of including the lab- and field work and the composition of the master dissertation in a ten-week Erasmus plus program in Zambia. What fortune I had to travel alongside an incredible team to the agricultural landscape of Zambia for three field interventions, to have a taste of real field work on a fascinating, though still problematic disease and to get to know the ever kind and helpful Zambian people and amazing people from around the world. My participation in the field study and the composition of the master thesis was made possible through financial support of Ghent University and the 2017-2018 Burroughs-Wellcome Travel Award. Furthermore, I would like to thank some particular individuals that contributed to its’ success. First of all, I have to thank my promotors, Prof. Dr. Sarah Gabriël and Dr. Kabemba. E. Mwape. They handed me the opportunity to cross continental borders to Zambia and made me feel most welcome at the University of Zambia and in the CYSTISTOP project. Thanks to them I was introduced in a most intruding subject that centers both human and porcine species. Furthermore I would like to thank them for all the time brainstorming, for providing scientific guidance and counsel and for the many corrections and adjustments they made. Secondly I want to thank Emma, Chiara, Lizzy, Chishimba, Chembe, Max and all the other field members for all the work we did, for the intense, crazy days and evenings we had and foremost, for your friendship. It wouldn’t have been the same experience without you all! Many thanks Zambia, your heartwarming inhabitants, your incredible nature and your ‘pole-pole’ style of life opened my eyes, pleased my senses and touched my heart. My stay and research work in Zambia were definitely one of the highlights of my veterinary studies. As the end of a chapter is approaching, I also want to thank my friends at home and my friends I met in Ghent, with whom I shared incredible moments in the last six years. I still remember a professor saying at the very beginning of our veterinary studies: “Please take time to look to your left and to your right. Next year only one third of you will be here”. I think the front and back row had a hard time, as all of us will be graduating this year and I am convinced that this is, apart from our own perseverance, due to teamwork, mutual support and motivation. Last but not least, I would like to conclude this word of thanks with the most important acknowledgement, being that of my parents. They endured and stood by me during six year veterinary studies, including some hysterical study outbreaks. Moreover, and more important, they support and believe in all my -sometimes irrational or out-of-the-box- undertakings. Thank you both for that, for everything else and for what is yet to come! Zikomo kwambiri

Table of Contents

Summary ............................................................................................................................................................................................... 6

Samenvatting ........................................................................................................................................................................................ 7

1. Introduction ....................................................................................................................................................................................... 9

1.1 Lifecycle, prevalence and burden of the parasite ..................................................................................................................... 9

1.2 Global interests ........................................................................................................................................................................ 10

1.3 Diagnostic challenges ............................................................................................................................................................. 11

1.4 Available control tools ............................................................................................................................................................. 12

1.5 Objectives ................................................................................................................................................................................ 15

2. Systematic Literature Review ......................................................................................................................................................... 16

2.1 Objectives ................................................................................................................................................................................ 16

2.2 Review Question ..................................................................................................................................................................... 16

2.3 Methods and Exclusion Criteria .............................................................................................................................................. 16

2.4 Results and discussion ............................................................................................................................................................ 17

A. Mathematical models ..................................................................................................................................................... 19

B. Human Treatment .......................................................................................................................................................... 25

C. Health Education ........................................................................................................................................................... 29

D. Improving Pig Husbandry .............................................................................................................................................. 32

D1. Vaccination .................................................................................................................................................................. 32

D2. Pig treatment................................................................................................................................................................ 34

D3. Sanitation ..................................................................................................................................................................... 35

E. Combinations ................................................................................................................................................................. 36

3. Field Study ...................................................................................................................................................................................... 38

3.1 CYSTISTOP project ................................................................................................................................................................ 38

3.2 Material and methods .............................................................................................................................................................. 39

3.2.1 Study area ................................................................................................................................................................. 39

3.2.2 Study animals ............................................................................................................................................................ 40

3.2.3 Human population ..................................................................................................................................................... 41

3.2.4 The elimination strategy ............................................................................................................................................ 41

3.2.5 Ethical considerations ............................................................................................................................................... 42

3.2.6 Post - elimination survey: Nyembe (E-arm) and Herode (N-arm) ........................................................................... 42

3.3 Results and Discussion ........................................................................................................................................................... 47

3.3.1 Coverage of the pig and human interventions ......................................................................................................... 47

3.3.2 Dissection results ...................................................................................................................................................... 49

3.3.3 Blood sample results (Eligible pig population) ......................................................................................................... 51

3.3.4 Stool sample results .................................................................................................................................................. 52

3.3.5 Impact of the intervention on the PCC prevalence and TS prevalence .................................................................. 53

4. Conclusion ...................................................................................................................................................................................... 56

References .......................................................................................................................................................................................... 59

Appendix 1. ......................................................................................................................................................................................... 65

Appendix 2. ......................................................................................................................................................................................... 66

Appendix 3. ......................................................................................................................................................................................... 67

Appendix 4. ......................................................................................................................................................................................... 68

Appendix 5. ......................................................................................................................................................................................... 69

Appendix 6. ......................................................................................................................................................................................... 69

List of Abbreviations

AFS: African swine fever C-arm: Control study arm CLTS: Community Led Total Sanitation DALY: Disability-adjusted life-year E-arm: Elimination study arm EITB: Enzyme-linked immunoelectrotransfer blot ELISA: Enzyme linked immunosorbent assay FAO: Food and Agriculture Organization GCLP: Good clinical laboratory practices GCP: Good clinical practices HCC: Human cysticercosis HE: Health education HH: Household ITFDE: International Task Force on Disease Eradication MDA: Mass drug administration MM: Mathematical model Mo.: Month(s) old N-arm: Negative Control study arm NBCS: Newborn calf serum NCC: Neurocysticercosis NCZ: Niclosamide NTD: Neglected Tropical Disease/ ‘Verwaarloosde tropische ziekte’ NZD: Neglected Zoonotic Disease OD: Optical density OPD: Ortho phenylenediamine OXF: Oxfendazole PBS: Phosphate buffered saline PCC: Porcine cysticercosis PCR: Polymerase chain reaction PE survey : Post-elimination survey PME: Post-mortem examination POC-test : Point-of-care test PRECEDE-model: Predisposing, Reinforcing, and Enabling Constructs in Educational Diagnosis and Evaluation model PZQ: Praziquantel SAC: School aged children SOP: Standard operating procedure SSA: Sub-Saharan Africa STH: Soil transmitted helminths/ ‘via de bodem overgebrachte wormbesmetting’ TCA: Trichloroacetic acid solution TS: Taeniasis UNEP: United Nations Environment Programme UNZA: University of Zambia WHO: World Health Organization

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Summary

The cysticercosis/taeniasis (TS) zoonotic complex, caused by the cestode parasite Taenia solium, is apart from a Neglected Tropical Disease (NTD) also the number one foodborne

parasitic disease (WHO/FAO, 2014). The complex disease, occurring in both pigs and human, is endemic in low and middle income countries, such as Zambia and is responsible for epileptic seizures, headaches, loss of life quality and high economic losses. Multiple strategies to tackle the problem are (commercially) available and information on existing or new strategies is rapidly evolving. However, a validated strategy is still lacking. Earlier, the suggestion was made that a combination of strategies tackling both human and pigs, combined with long-term measures is necessary to obtain a quick and sustainable reduction or elimination (Kyvsgaard et al., 2007). A large-scale study of this nature, proved effective in a Peruvian study (Garcia et al., 2016) but has never been carried out under Zambian conditions of transmission. The aim of this thesis was twofold. First, a systematic review was conducted to identify and review new (empiric) evidence regarding control and elimination of T. solium, published in the last three

years. Secondly, an evaluation of a two-year field study (CYSTISTOP), was carried out to assess whether the strategy succeeded in eliminating or rapidly reduce the parasite in pigs under the Zambian conditions of disease transmission. The systematic search resulted in the identification of 458 records of which we included 31. A relatively large number of mathematical models, confirmed once more that a combined approach is necessary to obtain a quick and sustainable impact on the prevalence of T. solium.

Besides, some new strategies using available tools were proven to be promising or efficient in a number of field trials. Among them, multiple round treatment, ring-treatment and treatment of TS cases incorporated in existing NTDs or soil transmitted helminths (STHs) programs. On the other hand, progress was also made in long-term strategies. Evidence-based health education tools (‘The Vicious Worm’) and methods (PRECEDE-PROCEED model) were developed and proven effective. In the CYSTISTOP field trial, significantly more T. solium positive pigs were found at dissection

in the Negative Control study arm compared to the Elimination study arm (OR=25; p=0.003) and an important reduction in occurrence of T. solium was achieved in the Elimination study arm (46% to 4%). As only one calcified, non-infectious cyst was found, elimination of active porcine cysticercosis was obtained. Protection by two consecutive vaccination shots was difficult to implement in Zambian rural areas, due to a sharp decrease in pig population (presumed due to African Swine Fever), and the high turnover in the pig population. Moreover, vaccination was more often refused by pig farmers in contrast to therapeutic treatment. Constrains identified during the field study combined with other potential constrains (storage and transport of the vaccine under appropriate cold chain conditions, availability of veterinarians…), make the efficacy of a combined vaccination/treatment scheme as suggested by Lightowlers and Donadeu (2017) less credible. However possible (Garcia et al., 2016; CYSTISTOP, 2018), elimination might be a too ambitious goal for low-and middle income countries, such as Zambia. These countries might, not yet, be ready to implement an economically and practically very demanding, intensive intervention. A stepwise approach, consisting of initial or regular therapeutic treatment, potentially implemented as a ring-strategy or integrated in an existing NTD/STH program, with additional long-term measures and active surveillance seems a more reachable strategy. Keywords: Taenia solium – taeniasis – cysticercosis – Neglected Zoonotic Disease – tapeworm – systematic review – intervention - control – elimination – sub-Saharan Africa – Zambia

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Samenvatting

Het cysticercose/taeniasis zoönotisch complex wordt veroorzaakt door de cestode parasiet, Taenia solium en behoort tot de ‘Verwaarloosde tropische ziektes’ (NTDs). Daarnaast is de

parasiet sinds 2014 nummer één op de lijst van voedselgebonden parasitaire ziekten (WHO/FAO, 2014). De complexe ziekte komt voor in zowel de humane als de porciene gastheer, meestal in landen met een laag tot gemiddeld inkomen, zoals Zambia. De parasiet is o.a. verantwoordelijk voor epileptische aanvallen, hoofdpijn, een verlies aan levenskwaliteit en hoge economiche verliezen. Verschillende strategieën waarmee het probleem kan aangepakt worden zijn (commercieel) beschikbaar en de kennis over bestaande en nieuwe mogelijkheden evolueert snel. Toch is een gevalideerde strategie nog steeds onbestaande. Kyvsgaard et al. (2007) suggereerde eerder dat een gecombineerde aanpak, waarbij zowel mens als varken behandeld wordt, in combinatie met lange-termijn maatregelen noodzakelijk is om een snelle en langdurige reductie of eliminatie van de parasiet te bekomen. Een grootschalige studie van deze aard was effectief in een Peruviaanse studie (Garcia et al., 2016), maar werd nooit eerder uitgevoerd onder Zambiaanse omstandigheden. Het doel van deze thesis was tweeledig. Ten eerste werd er een systematische review uitgevoerd, met als doel nieuwe (empirische) informatie over de controle en eliminatie van T. Solium, gepubliceerd

in de laatste drie jaren te identificeren en te beoordelen. Ten tweede werd er een evaluatie van een tweejarige veldstudie (CYSTISTOP) gemaakt om na te gaan of er d.m.v. de strategie een snelle reductie of eliminatie van de parasiet bereikt werd. De systematische zoekopdracht resulteerde in de identificatie van 458 artikels en boeken, van welke er 31 geïncludeerd werden. Een relatief groot aantal mathematische modellen bevestigde andermaal dat een gecombineerde aanpak noodzakelijk is om een snelle en aanhoudende impact te hebben op de prevalentie van T. solium. Daarnaast werden er een

aantal nieuwe strategieën geïdentificeerd, die efficiënt of veelbelovend bleken in een aantal veldstudies, waaronder meervoudige behandelingsronden, ring-strategieën en behandeling van lintwormdragers geïncorporeerd in bestaande programma’s voor NTDs of via de bodem overgebrachte wormbesmettingen (STHs). Daarnaast werd er ook vooruitgang geboekt wat betreft de lange-termijn maatregelen. Op wetenschappelijke kennis gebaseerde (evidence-based) educatieve toepassingen (‘The Vicious Worm’) en methoden (PRECEDE-PROCEED model) werden ontwikkeld en waren effectief in veldstudies. In de CYSTISTOP veldstudie werden er significant meer T. solium positieve varkens gevonden

tijdens de dissecties in de Negatieve Controle studie arm, ten opzichte van de Eliminatie studie arm (OR=25; p=0.003) en een belangrijke daling in het voorkomen van T. solium werd

verkregen in de Eliminatie studie arm (46% to 4%). Aangezien er enkel één gecalcificeerde, niet-infectieuze cyste werd gevonden, werd er eliminatie van actieve porciene cysticercosis bereikt. Bescherming d.m.v. twee opeenvolgende vaccinaties was moeilijk te implementeren in de landelijke gebieden van Zambia, gezien de sterke daling (vermoedelijk door Afrikaanse Varkenspest) en turnover in de varkens populatie. Daarnaast werd vaccinatie vaker geweigerd door de varkenshouders in tegenstelling tot medicinale behandeling. Limitaties opgemerkt tijdens deze veldstudie, in combinatie met andere mogelijke beperkingen (zoals bewaring en transport van het vaccin met inachtname van de geijkte koude keten, de aanwezigheid van voldoende dierenartsen,...) maken de efficaciteit van een gecombineerd vaccinatie- en behandelingsschema, zoals voorgesteld door Lightowlers en Donadeu (2017), minder geloofwaardig in deze omstandigheden. Hoewel eliminatie mogelijk is, zoals beschreven in de Peruviaanse en Zambiaanse studie (Garcia et al., 2016; CYSTISTOP, 2018), zou dit doel een té ambitieus kunnen zijn voor landen met een laag tot gemiddeld inkomen, zoals Zambia. Deze landen zijn mogelijks nog niet klaar om economisch en praktisch veeleisende en intensieve maatregelen te implementeren. Een stapsgewijze aanpak, die bestaat uit initiële of regelmatige behandeling, eventueel geïplementeerd als een ring-strategie of geïntegreerd in een bestaand NTD of STH programma, met bijkomende lange-termijn maatregelen en een surveillance systeem lijkt een beter haalbare strategie.

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Kernwoorden: Taenia solium – taeniasis – cysticercose – ‘Verwaarloosde zoönotische aandoening’ – lintworm – systematic review – interventie - controle – eliminatie – sub-Saharan Afrika – Zambia

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1. Introduction

1.1 Lifecycle, prevalence and burden of the parasite Though seen as an apparently remote health problem in Europe, Taenia solium infections can

cause significant health problems in endemic, mostly developing countries. The normal life cycle (Figure 2.) of the parasite involves the human final host and the pig intermediate host and causes three different diseases. A first disease occurs in the pig when it gets infected with the metacestode larval stage (cysticerc, porcine cysticercosis (PCC)) after ingestion of tapeworm eggs that are passed in the stool of tapeworm carriers. Humans are the only definitive hosts and become tapeworm carriers (taeniasis (TS)) after eating undercooked pork, infected with viable cysticerci. Health problems especially occur when humans become accidental intermediate hosts after ingestion of the eggs. The developing cysts then tend to localize in the central nervous system, causing human neurocysticercosis (NCC), a leading cause of acquired epilepsy in endemic areas (Murrell et al., 2005). Besides epileptic seizures, chronic headaches are a common symptom of NCC (Carabin et al., 2011).

The neglected parasitic zoonosis is most common in regions associated with poverty, a lack of sanitation and free-range pig husbandry, allowing direct contact with human faecal material. Moreover, laws on meat inspection and encouragement around safe consumption of meat are not (sufficiently) implemented (Murell et al., 2005; Kyvsgaard et al., 2007; Coral-almeida et al., 2015). Latin America, sub-Saharan Africa (SSA), South and Southeast Asia are currently the main endemic regions (Figure 1.).

Figure 1. Endemic regions of T. solium in 2015 (Figure: WHO,2016).

Recent studies in SSA show a prevalence of PCC varying from 2% to 41.2%, depending on the region and kind of diagnostic tool (Assana et al., 2013). In Zambia, where pig keeping and consumption heavily increased since 1990, the PCC prevalence varies from 7.3 to 34% (Phiri et al., 2002, 2003. Sikasunge et al., 2007, 2008). Studies in the Eastern province of Zambia show a prevalence of 6.3% -11% of TS (Mwape et al., 2012; Mwape et al., 2013), up to 20 % of PCC (Sikasunge et al., 2008) and 5.8% of human cysticercosis (HCC) (Mwape et al., 2012). Moreover, the prevalence of NCC among people with acquired epilepsy is reported to be almost 60%, which proves NCC to be the most important single cause of epilepsy in this area (Mwape et al., 2015).

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The public health issue was recently re-estimated1 as a leading cause of deaths from foodborne diseases, with a total of 2.8 million disability-adjusted life-years (DALYs) a year in low and middle income countries related to cysticercosis (WHO, 2015a). Besides, considerable economic losses in the public health and agricultural sector exists as well. These are mainly due to NCC, causing the loss of wage-earning activities (Urajkotia et al., 2007) and a reduced value of the infected carcasses. Annual losses due to PCC have been estimated at 25 million USD for ten West and Central African countries (Zoli et al., 2003) and 18.6 to 34.2 million USD for Eastern Cape Province, with on average 5 million USD attributed to agricultural losses (Carabin et al., 2006). At last an increasing number of human NCC cases is being detected within the European union and the United states, where T. solium is claimed to be eliminated,

primarily due to increased human migration (Serpa et al., 2012; Gabriël et al., 2015).

1.2 Global interests

Health issues and economic losses make clear that the need for the control and, if possible, elimination of the zoonosis exists. The first practical recommendations upon control of T. solium date back to 1976 (FAO/UNEP/WHO, 1977). Development of diagnostics and treatment

strategies have made a lot of progress in the past decades and the international communities’ interest and engagement has been stimulated. T. solium was declared ‘potential eradicable’

by the International Task Force on Disease Eradication (ITFDE) as early as 1993 (Schantz et al., 1993), but adjustments to this statement were made twice. In 2003, the need for a national scale pilot study as proof was highlighted (ITFDE, 2003). In 2013, the ITFDE acknowledged the existence of some challenges upon eradication, such as the lack of routine surveillance and reporting, the need for evidence of a financial benefit in better pig husbandry towards the conviction of farmers, the need for rapid diagnostic tests (see 1.3) and the need for data of how preventative chemotherapy affects prevalence (Center, 2013). These challenges are still remaining (Thomas, 2015). The World Health Organisation (WHO) highlighted T. solium cysticercosis as a Neglected

Zoonotic Disease (NZD) (WHO, 2006; WHO 2007a; WHO, 2010) and included the parasite in 2012 in the road map to tackle the Neglected Tropical Diseases (NTDs). This road map targeted a validated, stepwise approach for the control and elimination of T. solium, using the

most cost-effective control tools to be ready and available by 2015, with elimination interventions scaled up in selected countries from 2016 to 2020 (WHO, 2012; WHO, 2015b). The international community also pledged their commitment to this goal in the London Declaration (WHO, 2013) and World Health Assembly Resolution WHA66.12 also requested member states, international partners and the Director General WHO to provide support for the activities outlined in this road map. However, the 2015 goal has not been met (WHO, 2017) and up till now there is still no consensus about the most cost-effective and feasible approach for control (Lightowlers et al., 2016b). Though results of several studies and advices since 2012 should be considered and integrated to realize the targets of the roadmap. In 2014, the parasite was ranked first on the global scale of foodborne parasites (WHO/FAO, 2014) and in 2015, fourth on 31 investigated foodborne hazards (Havelaar et al., 2015). In 2015, a WHO informal consultation reporting the assembly of a framework for intensified control of TS and NCC caused by T. solium (WHO, 2015c) was held. This framework should

provide technical and operational support to countries for the initiation of large-scale control programs. Pilot programs on interrupting transmission of T. solium, improved case detection

and management of NCC with available tools are reported to be currently ongoing2 in six countries (personal communication S. Gabriël).

1 Earlier, Murray et al. (2012) estimated the global disease burden of 291 diseases including T. solium. 503,000 Disability-Adjusted

Life Years (DALYs) were related to cysticercosis. 2 To be found on: http://www.who.int/taeniasis/control/en/ (Last consulted in March 2017)

http://www.who.int/taeniasis/control/en/

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1.3 Diagnostic challenges

To carry out regular surveillance, to measure the impact of interventions or for the diagnosis of clinical cases in human, cost-effective, easy-to-use and inexpensive diagnostics that are suitable for large-scale implementation in resource-limited settings are needed, but lacking at the moment (WHO, 2016). Diagnosis of T. solium based on clinical symptoms is difficult as

they may be absent, mild, non-specific or occur in a late stage. In the case of HCC, people may develop chronic headaches, blindness, (epileptic) seizures or other brain-related symptoms and, in certain regions, may develop visible and most of the time palpable subcutaneous nodules. TS is mostly asymptomatic, symptoms include abdominal pain, nausea, diarrhea and other gastrointestinal symptoms (Murell et al., 2005). TS diagnosis can be performed based on microscopical stool examination, although the sensitivity is low and the test is not T. solium specific (Praet et al., 2013). Immunodiagnostic methods, such as the

copro-antigen enzyme linked immunosorbent assay (Ag ELISA) and copro-polymerase chain reaction (PCR) are more sensitive (84.5% and 82.7% respectively), and, especially the copro-PCR more specific (Mayta et al., 2008; Praet et al., 2013). The most optimal test for serological diagnosis of HCC is the enzyme-linked immunoelectrotransfer blot (EITB, 98% sensitivity, 100% specificity; Tsang et al., 1989). The two Ag ELISAs (HP10 and B158/B60) that detect T. solium cysticerci circulating antigens with

90% sensitivity and 98% specificity, respectively (Brandt et al., 1992; Praet et al., 2010b) are available, and often used for treatment follow-up. However, in cases of a single brain cyst or calcified cysts, the sensitivity of both antibody based tests has been shown to drop significantly (Chang et al., 1988; Singh et al.; 1999; Wilson et al., 1991), making imaging techniques such as magnetic resonance imaging and computer tomography necessary for a definitive diagnosis. Neuroimaging facilities are however lacking in most low-income countries and too expensive for local inhabitants. Moreover, PCR and EITB for the detection of both TS and/or HCC are expensive and need advanced laboratory equipment that might pose a challenge for low-income countries (WHO,2016). Currently, a lateral flow, point-of-care (POC) test to diagnose TS and NCC at the same time, developed by the Centres for Disease Control and Prevention in Atlanta, Georgia is being tested (Corstjens et al., 2014; personal correspondence with team member of the SOLID project3). This rapid and easy-to-handle diagnostic tests would also be able to pre-diagnose epileptic patients, where treatment with niclosamide (NCZ) instead of praziquantel (PZQ) and follow-up is necessary, given the possible side-effects of PZQ treatment (see 1.4). In pigs, diagnosis can be performed ante- or post-mortem. Tongue examination, by means of inspecting and palpating the tongue is a common diagnostic during purchasing, but the sensitivity of the method decreases when infection intensity is low (Dorny et al., 2004) (see 1.4). Circulating antigens can be detected by HP10 and the B158/B60 monoclonal antibody based Ag ELISA assays (Harrison et al., 1989; Brandt et al.,1992; Dorny et al., 2004). Both have shown high sensitivities (65 - 93%) and specificities (70–100%), however they are not commercially available and require a laboratory setting. Specificity can drop as the Ag ELISA cross-reacts with Taenia hydatigena (Devleesschauwer et al., 2013), resulting in false

positives and overestimation of PCC prevalence (Braae et al., 2015c). Sensitivity can drop if less or less viable cysts are present in the animal or carcass (Brandt et al., 1992; Dorny et al., 2004). Serological diagnosis of PCC by EITB has also been described, but showed, in contrast to the ETB fort the detection of HCC moderate sensitivity (89%) and specificity (43%) under field conditions (Yayashi et al., 2014). Meat dissection is specific, but the sensitivity is low if only few cuts are made and cyst loads are low (Dorny et al., 2004). Full-carcass dissections are considered the gold standard. However, these are only possible in research circumstances as the method is time consuming and the meat cannot be used for commercial purposes afterwards. In two recent studies a sensitivity of 81% (Lightowlers et al., 2015) and 71% (Chembensofu et al., 2017) was described after selective dissection of some predilection

3 https://www.ugent.be/di/vph/en/research/fpz/solid/ (Last consulted in February 2017)

https://www.ugent.be/di/vph/en/research/fpz/solid/

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places. At last, PCR can be used for the identification of suspected cysts (Gonzalez et al., 2006).

1.4 Available control tools

Available control measures can be divided into measures on human level, targeting the final host and measures on pig level, targeting the intermediate host. The final goal of any control measure is either a decrease in morbidity due to NCC, or a decrease TS and PCC prevalence (transmission), which eventually should lead to a decrease in NCC. All of these measures lower or prevent transmission of T. solium at one or more points in the life cycle (Figure 2.).

The first group, targeting the human final host, exist out of health education, hygienic and sanitary improvements and human treatment. Trials on health education (HE) are particularly interesting because the health risk of T. solium is not perceived as important due to the long gap between infection and clinical symptoms and the big attention to ill health and death directly related to other diseases such as Malaria and HIV virus. Trials on HE have previously shown to be effective, especially in terms of increased knowledge. However, HE as stand-alone approach does not guarantee the translation of knowledge in behavioral changes necessary for disease reduction (Sarti and Rajshekhar, 2003). HE is however considered essential and a combination with another strategy is suggested. Hygienic and sanitary measures can be found on two levels, the community level and the individual level. On the

one hand, the availability and use of latrines stops the parasite from being transmitted to the environment and free-roaming pigs and on the other hand, improved personal and household (HH) hygiene, made possible by the availability of clean water, hygiene and sanitation infrastructure, reduces the risk of NCC for the individual and his/her close contacts. The advantages and importance of sanitation measures reach beyond the goals for T. solium such

as other soil transmitted helminths (STHs) (Bethony et al., 2006) and diarrheal agents (Pruss-Ustun et al., 2014) and therefore fit in a broader One Health approach. A last measure on the human final host is treatment of tapeworm carriers. NCZ (efficacy: 85%) and PZQ (efficacy:

95 %) (Jeri et al., 2004; Rajshekhar, 2004) are the drugs of choice, destroying the direct sources of NCC. MDA as a stand-alone strategy was not always effective in the past (Lightowlers, 2013; Sarti et al., 2000), though studies on multiple rounds of treatment and new target groups (based on schistosomiasis co-endemicy or age group) have been executed recently (see 2.4, B.). A concern about PZQ is that the drug, unlike NCZ, is absorbed by the intestinal tract and possibly reaches and affects brain cysts in NCC infected people, which can result in adverse effects (seizures or headaches). These mainly occur when higher doses are used than the doses used to treat TS (5-10 mg/kg). This is for example the case for the treatment of schistosomiasis (40 mg/kg). Only one case reporting adverse effects after treatment with the taeniacidal dose has been reported in the past (Flisser et al., 1993). Though irrespective to the dose, sensitive, field-ready tests that detect NCC are needed to prevent adverse effects. Different treatment schemes are possible: drugs can be applied through mass drug administration (MDA), on the individual level based on diagnosis of TS (track and treat) or applied to certain risk groups. The latter, also called ‘focus group administration’ can be targeted to TS cases including close contacts or to TS cases within a certain risk area surrounding an infected pig (ring-treatment; see 2.4, B.). Strategies based on the diagnosis of TS cases are currently impractical, given the lack of sensitive detection methods as discussed in 1.3. Alternatively, triple doses of albendazole or mebendazole are also effective but might be impractical in the field. An advantage of albendazole and mebendzaole is however that these drugs also work against other STHs (Steinmann et al., 2011; Ash et al., 2015).

A second group of control measures, targeting the pig intermediate host includes meat inspection and proper meat cooking, pig husbandry and pig treatment and/or vaccination. Meat inspection could prevent infected meat entering the food chain and consequently prevent tapeworm carriers. Moreover, the idea that infected meat would be discarded might lead to better pig management practices, as one of the factors which influences behavior is the probability of economic consequences. However, up till now, only tongue palpations are done

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by farmers and traders and this method of low sensitivity is only able to identify highly infected animals (Dorny et al., 2004). Consequently, lightly infected pigs are still consumed and even the identified infected carcasses are still sold at the illegal market at a cheaper price or used for own consumption at the HH (Pondja et al., 2010; Praet et al., 2010a). Meat inspection as a control measure is currently not efficient due to these gaps. To improve, national legislation should be implected more strictly and on a lower level, consumer education would lead to a drop in the economical market of pig farmers, as consumers would be less inclined to buy infected pork. Moreover, a new, more sensitive meat inspection method should be implemented. Partial dissection of some predilection places could diagnose 71 to 81% of infected pigs in a recent study (Lightowlers et al., 2015; Chembensofu et al., 2017), suggesting this method to be an improvement on currently used methods. Awaiting better meat inspection, proper meat cooking can make infectious meat safe for consumption, as metacestodes are

destroyed when cooked at more than 45 to 50° within 15 tot- 20 min. (Cordero del Campillo, 1974; Flisser et al., 1986). By keeping pigs confined, they are prevented from eating human stool containing T. solium eggs. In this way, pig husbandry prevents PCC and interrupts the

life cycle of the parasite. Both positive (Gilman et al., 1999) and negative results have been found in studies on total confinement of pigs. A recent study by Braae et al. (2014b) found a comparable prevalence of PCC in confined pigs and free-roaming pigs, which suggest that also other factors have to be taken into account (such as feeding the pigs with waste food). Moreover, pig confinement is not an easy strategy to adopt. In a lot of endemic areas, free-roaming pigs are an available source of food or money for the family and way of keeping the premises clean without having to feed them. Confinement would undermine the economic benefit of keeping pigs for a lot of farmers (Thys et al., 2016); see 1.4, D3) To improve pig’s husbandry, cheap ways of pig housing and feeding are to be implemented. Confinement of pigs also prevents contact between different herds of pigs, lowering the occurrence of other infectious diseases such as African swine fever (ASF), often perceived important by farmers. Anthelmintic treatment of pigs with Oxfendazole (OXF, 30mg/kg) is another way to prevent pig meat to contain cysts. The drug was registered in 2013 in Morocco and registration in other endemic areas is currently ongoing4. OXF is cheap, easy to administer and effective against muscle cysts and other gastrointestinal helminths (Pondja et al., 2012; Mkupasi et al., 2013a; Mkupasi et al., 2013b), though ineffective against brain cysts (Sikasunge et al., 2008; Mkupasi et al., 2013b). A withdrawal period of 17 days is sufficient to guarantee safe human consumption (Moreno et al., 2012), but it takes up to six months for the cysts in the meat to completely resolve (Gonzalez et al., 1997; 1998; 2001; Sikasunge et al., 2008; Moreno et al., 2012). Immunization by vaccination prevents pigs from T. solium infection and can be a stand-

alone intervention or combined with OXF pig treatment. Different vaccines have been developed in the past (TSOL45, S3Pvac), but so far, TSOL18 has been the most promising and is the first commercially available vaccine, registered in India. Registration in other endemic countries is currently ongoing3. Studies on the vaccine showed a reduction of PCC prevalence (Jayashi et al., 2012) as a stand-alone intervention and eliminate PCC in the treated pigs if combined with OXF treatment (Assana et al., 2010). Although promising results, some practical issues upon implementation of the vaccine, such as the need for at least two vaccinations, the compliance of farmers, the maintenance of the cold chain and the cost of the vaccine in a poor area remain. At last, monitoring and surveillance programs are needed to prevent the parasite to

establish itself in non-endemic areas (Wandra et al., 2000; Gabriël et al., 2015) or to prevent the parasite prevalence to increase or re-establishes after the end of any control strategy (Kyvsgaard et al., 2007). Movement of people and pigs, possibly infected with T. solium should

therefore be registered and tested and/or treated to prolong any obtained effect. Moreover, these programs would render up-to-date prevalence and incidence data.

4 To be found on: http://www.who.int/neglected_diseases/news/First-licensed-vaccine-and-anthelmintic-against-epilepsy/en/

(Last consulted in March 2017)

http://www.who.int/neglected_diseases/news/First-licensed-vaccine-and-anthelmintic-against-epilepsy/en/

14

Yet considered an eradicable (Schantz et al., 1993) and tool-ready disease for many years, with various strategies proposed, only one elimination study in an endemic region has been carried out in with a positive, encouraging result. This was a large-scale, short-term, intensive program, tackling both human and pig host in Peru (Garcia et al., 2016). Another promising small-scale study in Cameroon, tackling only the pig host (treatment and vaccination) could reach elimination in treated pigs (Assana et al., 2010). Studies included in the Landscape analysis by Thomas (2015), a systematic review on control options, commissioned by the WHO, were executed in different regions, and often based on different single control options. Mostly these were only targeting one or two villages and did not include a control group. The intervention time, the follow-up and methods of monitoring differed as well. As a result, varying degrees of transitory reduction of the occurrence of the parasite were reported. Thomas (2015) reported that a decrease of transmission had been achieved through MDA to humans using NCZ or PZQ with and without the addition of HE or pig anthelmintic treatment. HE could cause a reduction in transmission in some studies, although it was difficult to prove that the reduction was a direct result of the interventions used. At last, a combination of OXF and vaccination could treat and prevent PCC although efficacy on the prevalence of human TS and cysticercosis remained to be determined. Integrating these results of the systematic review by Thomas (2015) and the outcome of mathematic model by Kyvsgaard et al. (2007), a long-term or integrated strategy, covering both human and pig hosts, supplemented by long-term measures (HE, improvement of sanitation, pig husbandry, meat inspection…) seems necessary to reach elimination or sustained control, as each of the control strategies have strengths and limitations. The latter was also recommended by the WHO ( 2014). Yet, the optimal treatment and/or long-term intervention combinations, detailing the frequencies and target groups in terms of efficacy and cost-efficiency needs to be determined.

Figure 2. Life cycle of T. solium and possible interventions (X) which are considered to interrupt the life cycle in endemic areas.

15

1.5 Objectives

A ten-week master student-exchange between the Department of Veterinary Public Health and Food Safety (Ghent University, Belgium) and the Laboratory of Clinical studies (University of Zambia (UNZA), Zambia), participating the operational activities of the CYSTISTOP project (see 3.1) led to the final master dissertation including following objectives:

- To conduct a systematic review and identify new (empiric) evidence regarding control and elimination of T. solium since the latest review (Thomas, 2015).

- To determine whether elimination or rapid reduction of the parasite in pigs is possible under the Zambian conditions of disease transmission, through a short-term integrated strategy, targeting both humans and pigs.

16

2. Systematic Literature Review

2.1 Objectives

The general objective of this systematic review is to identify and summarize the recent evidence in English scientific literature on the control and elimination of T. solium since the systematic review by Thomas (2015). A first objective is to identify and collect all relevant articles published after 01/01/2014, including empirical data as well as evidence relating to aspects of NTD control which are directly relevant to the control and elimination of T. solium. A second objective is to critically assess these publications on the new evidence on existing and new strategies for the control and elimination of T. solium. The assessment will be based

on the type of intervention, the study population and coverage, the outcome variables and the presence of a follow-up period, randomization and a control group. Also non-empiric, supporting or non-supporting data will be taken into account during this assessment. As a result, this review will render a complete and actual overview of the recent accessible information and evidence on the control and elimination of T. solium and will give, combined with the review by Thomas (2015), a full picture on existing strategies and their effect. The review will therefore contribute to the rapidly evolving field of evidence on control and elimination of T. solium. 2.2 Review Question

‘Is there new evidence on the efficacy and development of existing or new control tools on the control or elimination of T. solium since 2014?’ Identifying key elements of the question using the PICOT acronym:

- Population: humans or pigs - Intervention: drugs (Praziquantel, Nicosamide, Albendazole, mass drug

administration, TSOL18, vaccination, oxfendazole), education, latrines, sanitation, husbandry or other

- Comparator: non-treated, local/experimental study population - Outcome: efficacy, side effects, acceptance, costs, risk factors - Timeframe: From 2014/01/01 till 2017/05/01, supplementary articles till 2018/03/01

2.3 Methods and Exclusion Criteria

A literature search was realized using similar methods as in the systematic review by Thomas (2015). Consequently, conclusions of this review will be complementary. The search was performed in twofold by two independent researchers by the following procedure:

- Following search engines were used: IngentaConnect, PubMed, Library of Congress, British Library, ScienceDirect, African Journals Online and Google Scholar.

- Following combination of search terms was used: 2014/01/01 (date of publication) AND Taenia solium OR cysticercosis OR taeniasis AND control OR elimination

- Searches were performed within titles and abstracts.

Afterwards, duplicates were removed and the articles were screened first on title, secondly on abstract and finally on full text. Exclusion of non-fitting papers were made according to the following criteria:

- Studies published before 01/01/2014 - Studies not relating to humans or pigs - Studies not relating to NTDs - Studies on aspects of NTDs which DO NOT discuss issues relevant to T. solium

control/elimination (e.g. no control intervention) - Papers relating to clinical symptoms, diagnoses and treatment of NCC including

case studies

17

- Purely epidemiological studies on T. solium - Papers on diagnoses of T. solium cysticercosis/ TS (including diagnostic imaging)

- Papers on aspects of basic sciences (immunology/ molecular biology/ physiology/ bio chemistry/ basic pharmacology)

- Study’s about control/ elimination in Europe/ US - Paper not written in the English language - Review

At last additional resources were identified by accessing citations within selected papers and by implementation of articles published after the initial search. 2.4 Results and discussion

The database search entry resulted in the identification of 458 records of which we included 31 after removing the duplicates, screening the titles, abstracts and full texts, based on the above mentioned exclusion criteria and including additional records. Of the 31 papers included in this analysis, 13 are field trials relating to the control of T. solium. Eighteen other articles contain experimental (non-field) data, data relating to aspects of NTD control that were directly relevant to the control of T. solium, meeting reports and national/international guidelines and strategies. (Figure 3.). Studies included in the review are underlined in References. Field and

experimental studies are included in tables classified by type of intervention.

18

Records identified through database

searching

(n = 458)

Scre

enin

g

Inclu

ded

E

ligib

ility

Id

entification

Additional records

identified through other

sources (n = 4)

(n = 4)

Records after duplicates removed

(n = 308)

Records screened on title

(n = 308)

Records excluded on title

(n = 160)

Abstracts excluded

(n = 85)

Full-text articles assessed for

eligibility

(n = 67)

Studies included in

systematic review

(n = 31)

Duplicate records excluded

(n = 150 )

Abstracts assessed for

eligibility

(n = 148)

Full-text articles excluded

(n = 36)

Figure 3. Flow chart Diagram

19

A. Mathematical models

Data characteristics

Simulation duration

Estimated prev. / P of infection

Population size Outcome Limitations mentioned in the article Citation

Latin America 4 y. - TS mean prev.: 1.5 - 5% - PCC mean prev.: 30 - 40%

- 1000 pp., 200 pi. - TS and PCC prev. - Lack of inclusion of environmental factors/age- and social structure. - Efficacy of drugs/vaccines set unrealistically high.

(Johansen et al., 2017)

Tanzania 4 y./ till burn

out

- 3.0% TS

- 13% PCC

pp. and pi. based on the

2012 census data from 2007/2008 agricultural

census data of Mbeya/ Mbozi district.

- P of elimination

- Time to elimination - TS and PCC prev.

- Lack of inclusion of spatial structure/ clustering effect

(if population bigger, chances of elimination smaller)/ influx of potential carriers in the system/ increased slaughter rates due to ASF mortality and sales of pigs.

(Braae et al.,

2016a)

Endemic areas 13 m. X X Risk that age cohorts of animals have the potential to transmit T. solium.

- Ignores the risk of animals that were not included in the interventions, or that were imported from areas outside the intervention zone, presenting a risk for transmission of T. solium.

(Lightowlers and Donadeu al., 2017)

Western Kenya 1 y. 0.376 (0.238±0.513) 230,253 pp., 21,315 pi. (76.0% pi.- consuming)

- Risk of a pork meal containing a potentially infective T. solium cyst at

the point of consumption. - Potentially infected meals a year

- Value of meat inspection

- Not each potentially infectious consumed meal will lead to a case of TS. - Assumption of evenly distribution of cysts in the muscles, while un-even distribution is more realistic.

- Not sure whether the proportion of pigs falling into infection intensity categories based upon the results from pigs of Zambia, is representative for Kenyan pop.

- P of a cyst to be viable is difficult to estimate.

(Thomas et al., 2017)

sub-Saharan

Africa

20 y. - 2% TS

- 7% HCC - 20% PCC

- 10000 pp., 2000 pi. - Cumulative number of HCC cases

averted during the intervention - HCC, TS, PCC prev. - Nr. of eggs in the environment

- Does not include the development of NCC (lack of

data). - Lack of inclusion of age structure/ heterogeneity of risk in pp. /spatial or seasonal elements.

(Winskill et al.,

2017)

Table 1. Summary of the characteristics of recent mathematical models. (ASF: African Swine Fever, m.: month(s), nr.: number, P: probability, PCC: porcine cysticercosis, pi.: pigs, pop.:

population, pp.: people, prev.: prevalence, TS: taeniasis, X: no results for this variable, y.: year(s))

20

Mathematical models (MMs) can be obtained in a quick way, are cheap to implement and render quick theoretical insights in which intervention tool, which algorithm or which stepwise approach of interventions will prove most useful in obtaining control or elimination. Five MMs, published since 2014 were included in the systematic review (Table 1. and 2.). Three of them

focus on single intervention options and combinations thereof targeting the human populations as well as the pig intermediate host. The fourth modelled the outcome of three interventions targeting the pig population. A last model assessed the risk of eating pork meat in western Kenya and the effect of meat inspection on the outcome.

The aim of the MM published by Johansen et al. (2017), was to calculate the theoretical outcomes of various control options suggested for TS and PCC elimination SSA (mass treatment of humans, vaccination and treatment of pigs, HE to communities and an integrated One Health approach) over a 4-year period. The model was built on the Reed-Frost transmission model, developed by Kyvsgaard et al. (2007), which is based on data from Latin-America. However, epidemiological data from SSA, published afterwards, fit the model well. A reduction of PCC or TS of at least 50% was calculated for all strategies, but they failed to maintain or improve this reduction. Short, single control options and even an integrated One Health approach were not able to obtain sustained control or elimination. Based on the results, elimination may be an unrealistic goal and thus, the suggestion was made to replace elimination into realistic determined control goals (reduction in morbidity (NCC) or reduction in transmission (PCC or TS)). Furthermore, the author highlights that there is a need to develop surveillance strategies since two earlier T. solium models predict a return to baseline levels

after stopping the control programs (Gonzalez et al. 2002; Kyvsgaard et al. 2007).

The deterministic, compartmental transmission EPICYST model (Winskill et al., 2017) is another MM build on the Reed-Frost model of Kyvsgaard et al. (2007). HCC prevalence and averted cases, as well as TS and PCC prevalence and number of eggs in the environment were calculated by deriving the basic reproduction number, R0. Outputs for six single interventions (two pig-focused interventions: vaccination and MDA; three behavioral/ infrastructural interventions: improved animal husbandry, improved sanitation and improved meat inspection; as well as tracking and treating tapeworm carriers (track and treat)) and some combinations thereof were modelled. All single interventions had the ability to reduce the prevalence of TS and HCC, the number of eggs in the environment and the prevalence of PCC. Chemotherapeutic interventions in humans or pigs would be the most effective (human track and treat, MDA and vaccination of pigs respectively from highest to lower effect) compared to improved animal husbandry, improved sanitation and improved meat inspection. Combinations of the human track and treat intervention resulted in small marginal gains. Combinations of behavioral and/or pig-targeted interventions were modelled to have considerable gains in effectivity. A single strategy of human track and treat was calculated to be most effective but is currently still a hypothetical intervention, made under the assumption that in the future more optimal diagnostics will be available to identify tapeworm carriers. Up till now, barriers exist to the introduction and financing of such an intervention on a large-scale, as potential adverse effects can occur when applying certain chemotherapeutics to NCC patients (see 1.4). Alternatively, pig MDA and pig vaccination can reduce prevalence of HCC in a short period, however a high coverage and efficacy rate is necessary when applied annually. In settings where fulfilling these two conditions is unlikely, a benefit can be obtained by a combination of pig MDA and pig vaccination with each other or with any of the other interventions. However, marginal gains of combinations are modelled if pig-focused interventions are implemented with sufficient coverage and efficacy rates. Improved animal husbandry, sanitation and meat inspection had less pronounced effects when applied as a single strategy, but some combinations can obtain effects similar to the single application of track and treat or pig MDA. Moreover, wide public health benefits on diarrheal diseases (Fewtrell et al., 2005) and other helminth infections (Ziegelbauer et al., 2012) make these options potentially more cost-effective.

21

A third model is the first agent-based CystiSim model and was developed by Braae et al. (2016a). This adaptable model uses field data from Tanzania and evaluates three intervention measures or combinations thereof: human treatment, pig treatment and pig vaccination. Elimination probabilities of PCC and TS prevalence in ten four-year intervention programs and time to elimination in five elimination interventions were calculated. This MM too, predicts that control is possible by targeting only the porcine host and again, efficacy and coverage are pointed out as limiting factors when it comes to elimination. In a realistic setting, where the pig coverage is below 90%, anthelmintic treatment combined with vaccination or inclusion of an additional human targeting intervention is a more robust strategy. Combinations targeting both hosts, have a high probability of success if a coverage rate of 75% can be maintained and are therefore the optimal strategy for obtaining elimination in the shortest time span. However, a combination of MDA to the whole human population and pig treatment would take a lot of resources to maintain a coverage above 75%. A coverage rate of 90% in school aged children (SAC) is a more realistic number, as it is more easy and practical to localize and treat them compared to adults. Similar results are however seen for a stand-alone pig treatment strategy, if a coverage rate of 90% can be obtained. Human MDA as a single approach was inadequate in terms of elimination, but might be a good strategy for controlling T. solium when carried over

an extended period. Although, this would depend on the cost-effectiveness and safety of this intervention. In contrast to the MM of Johansen et al. (2017) and Kyvsgaard et al. (2007), CystiSim includes parasite maturation, host immunity, and environmental contamination. When comparing to the results of Johansen et al. (2016), CystiSim scenarios are more likely to succeed, more intensive and have a more realistic approach to coverage and efficacy. Another difference is the speed at which TS and PCC returns to pre-intervention levels. CystiSim predicts a slower increase in prevalence compared to the model by Kyvsgaard et al. (2007) after the end of any intervention program. If this is correct, discontinuation of a four-year control program in SSA could have a greater impact than expected. The two models predict similar outcomes when interventions are implemented as single interventions. Both MMs also predict that control is possible, but elimination might be reachable.

The many options available for reducing T. solium transmission (Figure 2.) encourage the adoption of a multidisciplinary approach. Though, the implementation of multiple control measures in poor endemic areas might be more expensive and complex than adopting a minimal strategy. According to Lightowlers and Donadeu (2017), a minimal intervention strategy targeting only the pig intermediate host, might be a more sustainable and practical approach as fewer restrictions apply to pigs concerning ethics and implementation as compared to humans. Evidence already exists that such a strategy would reduce the incidence of human NCC in two or three years (Lightowlers et al., 2010). In the logical model presented by Lightowlers and Donadeu (2017), the efficacy of pig interventions, involving either the combined use of vaccination and OXF treatment or OXF treatment alone is predicted. Three-monthly OXF treatment to pigs >2 and <7 months old (mo.) alone is not able to prevent PCC in the whole population, resulting in a potential risk of transmitting T. solium in pigs of slaughter

age. Even when the withholding period of 21 days to pigs of slaughter age is not taken into consideration, this remains the case. The combined use of vaccination (>7mo.) and OXF treatment (> 2 and <7 mo.) presents a more effective strategy. After ten months, the intervention would be fully established and pigs older than six months of age that had at least two vaccinations and OXF treatment would not present a risk for transmission anymore. Two intervention strategies involving simultaneous application of treatment and vaccination in respectively older and younger pigs stand out to have the potential to prevent T. solium

transmission in pigs at slaughter age. Neither of them involves treatment of pigs at slaughter age. One intervention is the three-monthly vaccination plus OXF treatment cycle as modeled in the publication. The other is an intervention program where the animals receive two immunizations and OXF treatment, given one month apart, with a six-month interval. In contrast to Braee et al. (2016), a four-monthly based pig treatment (> 2 and <7 mo.) and vaccination (>7mo.) progam would not be able to prevent T. solium from being transmitted as 50% of the pigs at slaughter age would still be capable of transmitting the parasite. However,

22

the models are difficult to compare as they have a different set-up and different outcome measures are calculated. By implementing strategies, suggested in the article, HCC would drop within two or three years (depending on the eggs present in the environment). For a rapid impact on human-to-human transmission the authors suggest a single MDA round, timed after an effective intervention in the pig population. Coverage was previously mentioned to be an important factor when it comes to pig treatment and vaccination. Lightowlers and Donadeu (2017) did not mention the suspected coverage of a combined pig treatment/vaccination program. The question remains whether a coverage of 75% (as determined by Braae et al. (2016a)) of the pig population is feasible, given the possible outbreaks of pig diseases, the pig import from outside areas, the free-roaming pigs which are not easy to catch and uncooperative pig owners.

At last, a stochastic risk assessment model using Monte Carlo simulation was built using the @Risk add-on for Excel by Thomas et al. (2017). This model revealed that any pork meal consumed in western Kenya has a 0.006 (99%; 0.0002±0.0164) probability of containing at least one viable T. solium cysticercus at the point of consumption and therefore being potentially infectious to humans. This result also equates to 22,282 (99%; 622±64,134) potentially infective pork meals consumed in the course of one year within Busia District alone. Moreover, meat inspection, as currently practised in western Kenya was modelled to avoid only 1,397 (99% U.I. 5±8,368) potentially infective meals a year. Possible explanations include the significant underestimation of infected carcasses by methods used in slaughter houses, such as tongue palpation and meat inspection (Phiri et al. 2003; Dorny et al. 2004; Nsadha et al., 2014) and the presence of an illegal market where infected meat is sold at a lower price.

STRATEGY Control

(reduction TS

and PCC prev.)

Elimination (yes/no, P, time to

elimination)

Reduction in transmission

Citation

HU

MA

N T

RE

AT

ME

NT

Single SAC MDA (PZQ 40 mg/ kg, 90 to

100% cov. and efficacy).

Yes: 2 to 1% (TS) , 20 to 15 % (PCC)

after 3m.

No X (Johansen et al., 2016)

Human MDA annually

(4y, 90% efficacy, 75% cov.).

Yes 0.00 X (Braae et al.,

2016a)

SAC MDA annually (4y, PZQ 40 mg/ kg, 90 to 100% cov. and efficacy) (1).

Yes: 2% to 1% (TS), 20 to 11% (PCC).


SAC MDA annually (4y, PZQ 40 mg/ kg,

90% efficacy, 90% cov.).

Almost no effect. No X (Braae et al., 2016a)

MDA (PZQ 40 mg/ kg)

annually (90% efficacy, 90% cov.).

Yes 26 (10±90) y. X (Braae et al.,

2016a)

Annual human test

and treat (90% cov., 99% efficacy).

Yes X Median of 94% averted

HCC cases (95% CI: 83–97%).

(Winskill et al.,

2017)

PIG

T

RE

AT

ME

NT

/VA

CC

INA

TIO

N

(SIN

GL

E)

Pig tx., annual (4y.,

OXF, 90% efficacy, 75% cov.) (2).

Yes: 2 to 1% (TS),

20 to >7% (PCC)

No X (Johansen et al.,

2017)

Annual tx. of infected pigs (90% cov., 99% efficacy).

Yes X Median of 74% averted HCC cases (95% CI: 59–80%)

(Winskill et al., 2017)

Pig tx., 4-monthly (4y., OXF 30mg/kg,

90% efficacy, 75 or 90% cov.).

Yes 0.89 (90% cov.) 0.62 (75% cov.)

X (Braae et al., 2016a)

Table 2. Summary of the outcome of different strategies included in the mathematic models summed up in Table 1. (CI: confidence interval, cov.: coverage, HCC: human cysticercosis, m.: month(s), MDA: mass drug administration, mo.: month(s) old, OXF: oxfendazole, P: probability, PCC: porcine cysticercosis, prev.: prevalence, PZQ: praziquantel, SAC: school aged children, TS: taeniasis, tx.: treatment, U.I.: unit interval, vacc.: vaccination,

X: no information of this variable, y.: years, *: combination of (1), (2), (3) and (4))

23

Pig tx. (OXF), 4-

monthly (90% efficacy, 75% cov.).

Yes 49 (24±119) m. X (Braae et al.,

2016a)

Pig tx. (OXF), 3-

monthly >2 and <7 mo.

X X Majority of pigs remain

capable of transmitting during part of tx. interval.

(Lightowlers and

Donadeu, 2017)

Pig tx. (OXF), 3-

monthly >2mo.

X X Majority of pigs remain

capable of transmitting during part of tx. interval.

(Lightowlers and

Donadeu, 2017)

Annual pig vacc. (84% cov., 99% efficacy).

Yes X Median of 68% averted HCC cases (95% CI: 52–73%).


PIG

TR

EA

TM

EN

T/V

AC

CIN

AT

ION

(C

OM

BO

)

Pig vacc. and tx, 6- monthly (4y., 1-9 mo. pigs (80% of pop.,

75% cov., 100% vacc. efficacy, 90% tx. efficacy) (3).

Yes: 20 to <1% (TS), 20 to <5% (PCC)


Pig vacc. and tx, 4- monthly (4y., OXF 30mg/kg, 90%

efficacy, 75 or 90% cov.).

Yes 0.98 (90% cov.), 0.83 (75% cov.)

X (Braae et al., 2016a)

Pig vacc. (> 7 mo.)

and tx. (OXF), (>2 and <7 mo.), 4-monthly.

X X 50% of pigs> 7 mo. at risk of transmitting T. solium.

(Lightowlers and

Donadeu, 2017)

Pig vacc. and tx.

(OXF), 4-monthly (90% efficacy, 75% cov.).

Yes 42 (20±100) m. X (Braae et al.,

2016a)

Pig vacc. (> 7 mo.) and tx. (>2 and <7

mo.), 3-monthly .

X X Prevention of transmission in all animals >6 mo.

(Lightowlers and Donadeu, 2017)

CO

MB

INA

TIO

N H

UM

AN

/PIG

TR

EA

TM

EN

T (

& L

ON

G-T

ER

M S

TR

AT

EG

Y)

Integrated cross-sectoral approach

(4y) *.

Yes, highly significant effect


Human MDA (PZQ 40 mg/ kg) annually + pig tx. (OXF) 4-monthly

(4y, 90% efficacy, 75% cov.) (4y)

Yes 0.90 X (Braae et al., 2016a)

SAC MDA (PZQ 40

mg/ kg) annually + pig tx. (OXF) 4-monthly (4y., 90% efficacy,

75% (pig) or 90% (human) cov.).


2016a)

Human MDA annually

(PZQ 40 mg/ kg), 4 y. + pig vacc. and tx. (OXF) 4-monthly (4y,

90% efficacy, 75% cov.).


2016a)

SAC MDA (PZQ 40

mg/ kg) annually, 4 y. + pig vacc. and tx. (OXF) 4-monthly (4y,

90% efficacy, 75% (pig) or 90% (human) cov.).


2016a)

MDA (PZQ 40 mg/ kg) annually + pig vacc.

and tx. (OXF) 4- monthly (90% efficacy, 75% cov.).

Yes 40 (19±100) m. X (Braae et al., 2016a)

SAC MDA (PZQ 40 mg/ kg) annually + pig vacc. and tx. (OXF) 4-

monthly (90% efficacy, 75% (pig) or 90% (human) cov.).

Yes 32 (13±72) m. X (Braae et al., 2016a)

Annual pig tx. and human track and treat

Yes X Above a median of 75% averted HCC cases.


24

TS cases (90% cov.,

efficacy=99%). H

EA

LT

H

ED

UC

AT

ION

Health education, 1x (4).

X X 50% after 3m. (Johansen et al., 2017)

ME

AT

INS

PE

CT

ION

Meat inspection (as

currently practiced in western Kenya).

X Xx Avoids 1,397 (99% U.I.:

5±8,368) out of 22,282 potentially infective meals a y.

(Thomas et al.,

2017)

Meat inspection. Yes X Median of 56% averted HCC cases (95% CI: 40–71%)]


SA

NIT

AT

ION

(OR

CO

MB

O)

Improved sanitation. Yes X Median of 54% averted HCC cases (95% CI: 32–

77%)


Sanitation and inspection.

Yes X 78% (95% CI: 65–87%) (Winskill et al., 2017)

Sanitation and Pig treatment.

Yes X Above a median of 75% averted HCC cases.


HU

SB

AN

DR

Y Improved animal

husbandry.

Yes X Median of 41% averted

HCC cases (95% CI: 22–62%),

(Winskill et al.,

2017)

Though a quick and cheap method of rendering information, MMs have some limitations. Firstly, MMs run on the available biological/epidemiological data of a determined region. This leads to the fact that the published outcomes of MMs are valid only for the modelled area or for areas with a comparable setting. In the latter scenario, results should be interpreted carefully. For areas with a different set-up, area-specific data should be entered in the model, which might not be possible if the models are not free to use. Evidently, these data should be known, so epidemiological or biological studies should be conducted before intervention strategies can be modelled. Secondly, we can deduct from the results of this review that some interventions have different outcomes, depending on the model. It is difficult to compare results from different MMs as they use different mathematics, include other or more factors and make other assumptions for unknown parameters. Though it might be interesting to compare results of different models to prepare an intervention and gather an idea of the possible factors that might have an impact on the outcome of the intervention. At last, data on the contribution of environmental factors to the transmission of T. solium is still lacking. For example, information

on the survival of eggs in the environment, the contribution of flies and other insects or the life span of the tapeworm can either not be included or has to be assumed in the models. In conclusion, MMs remain simplification of the reality with sill a lot of unknowns, which are no catered for in the model. As such, MMs are a useful tool, but outcomes should be interpreted carefully and field trials are still the golden standard to obtain valid results of the efficacy of any intervention

25

B. Human Treatment Table 3. Summary on the recent field trials involving human treatment. (ALB: albendazole, BL: baseline, CI: confidence interval, cov.: coverage, d.:day(s), HH: household, MDA: mass drug administration, m.: month(s), NCZ: niclosamide, PCC: porcine cysticercosis, pi.: pigs, pp.: people, PZQ: praziquantel, R: treatment round SAC: school aged children, STH: soil transmitted helminths, TS: taeniasis, X: no results of this variable, y.: years, ≠: difference, ♂: male). The two articles indicated with * were written about the same field study.

Country, region

Year Population size/coverage

Intervention Incidence1 / Prev.2 Risk factor Follow-up period

Random Control study arm

Citation

TS PCC

Peru, Surpampa and Santa Ana

? 1,058 pp. (Surpampa), 753 pp. (Santa Ana).

4-monthly ring-screening and tx. (NCZ) TS cases, 100 m. from heavily-infected PCC pi.

Adjusted prev. TS at 16m.: ± 4 x lower in the intervention arm compared to control at 16 m.(prev. 0.28, 95% CI 0.08–0.91).

-Intervention-arm: 41% reduction (12 m.) compared to BL (incidence rate ratio 0.59, 95% CI 0.41–0.87). -Control: unchanged.

X 4, 8, 12, 16 m. Selected on similar size/terrain/ visible presence free-range pi.

Yes (Santa-Anna)

(O'Neal et al., 2014)

Tanzania, Mbozi and Mbeya district.

2012-2015

14 villages, 1500 pp., 400 pi. per district, per survey.

- Annual MDA rounds to SAC (2 Mbeya, 3 Mbozi): PZQ (40 mg/kg). - Track and treat of TS cases (NCZ, total sampled population).

- Mbozi: prev. adults 4,1 % to 1,8% (p=0.031); prev. children: 2.3% to 0.1%. - Mbeya: no significant result.

- Mbozi: 13% to 8% (p=0.002) Mbeya: no significant result.

- Children: decreasing age (p<0.001) (Mbeya). - Adults: increasing age (p=0.026), being ♂ (p<0.028, Mbozi). - Age of the pi. (p<0.001, Mbozi).

BL, 3 (pi. only), 6 12, 24 and 32 m.

X No (Braae et al.,

2016b)*

Tanzania, Mboziand Mbeya district.

2012-2014

14 villages, 305,319 pp., (Mbeya), 446,339 pp. (Mbozi) and 31,190 pi. (Mbeya) and 117,483 pi. (Mbozi). MDA cov.: 34 % of total population. Track and treat cov.: 9% of TS cases.

SAC MDA, 1 (Mbeya) or 2 (Mbozi, annual) rounds (PZQ, 40 mg/kg), track and treat TS cases (NCZ, total sampled population).

R0: ≠ between Mbozi (3%) and Mbeya (1.5%) (p=0.007) R1: - Mbozi: 3% to 2% (p = 0.024). - Mbeya: 1,5 to 0.3%, (p=0.004), ≠ between two disricts (p<0.001). R2: - Mbozi: 2% to 0.8% (p < 0.001) - Mbeya: 0.3%, to 0.5% (p=0.051), no ≠ between two districts (p=0.51).

X - Being ♂ (p=0.004). - Being adult compared to SAC. (p<0.001) - Living in Mbozi (p<0.001).

BL, 12 m. and 22 m. after first MDA.

Selected by knowledge/PCC presence.

No (Braae et al,

2017)*

Southeast Asea, Lao PDR

Nov. ‘13- Apr.

‘14

298 and 295 pp., 60 HHs, 64% cov.

2 MDA rounds (ALB 400 mg, 3 d., interval of 5

m.).

- TS decrease by 79.4% (MDA1). - prev. steady during inter-

treatment interval - 100% decrease (MDA2).

STH X BL, 1 m. and 5 m. after MDA 1. 1 m. after MDA

2.

No No (Ash et al., 2015) - prev. decreased by

65.5% (MDA 1) and 62.8% (MDA 2). - Increase during the inter-treatment interval. - Infection intensity decreased.

26

When the WHO included T. solium cysticercosis as a NTD in 2010, MDA was recommended

as the primary intervention strategy against TS (WHO, 2010). The systematic review of L.F. Thomas (2015) concluded that reduction of transmission over a short period is achievable through human MDA (using NCZ of PZQ) as a single or a combined control option. Recent mathematic models (see 2.4, A) suggest that a single round of human treatment is not sufficient to obtain sustained control, but a multiple round MDA schedule or a combined approach is necessary to obtain this goal (Braae et al, 2016a; Johansen et al., 2017; Winskill et al., 2017). Therefore, the scope of interest has recently moved towards finding a combination and algorithm of multiple strategies (see 2.4, E) and all recent field studies on single human MDA (Table 3.) included multiple treatment rounds. Two recent field trials (Braae et al., 2016b; 2017; Ash et al., 2015) studied the potential of integrating T. solium control into larger NTD MDA

programs and were included in this review. These strategies fit in a larger One Health approach as they have the potential to tackle more diseases at once and might improve cost-benefit ratios. One of these studies (Braae et al., 2017) compared a one and two round MDA treatment schedule, supporting the multiple round suggestion of MMs with actual field data. The group of O’Neal et al. (2014) is currently developing a focus based treatment protocol (ring-strategy) and published the first data on this strategy concerning the proposed treatment area and the efficacy of human focus based treatment. Further studies on the efficacy of this strategy (including a combined human-pork ring-strategy) were reported to be ongoing and data on cost-effectiveness are to be determined. Two articles were published on the field study by Braae et al. (2016b, 2017), the first large-scale TS control program implemented in SSA. One article assessed the effect of the national schistosomiasis control program on the prevalence of TS and PCC (Braae et al., 2016b). In a second article, the effect of MDA of PZQ in SAC in combination with track and treat of TS cases and the significantly better effect of repeated MDA was confirmed (Braae et al. (2017). SAC MDA (PZQ, 40 mg/kg) was carried out, either in two and three annual rounds, based on the schistosomiasis prevalence (Braae et al., 2016b), or in one and two annual rounds (Braae et al., 2017). In both articles, treatment was combined with additional track and treat of TS cases in the whole sampled population based on a copro-Ag ELISA test. In the first article Braae et al. (2016b), a significant decrease of TS and PCC was found after three rounds, whereas a two-round MDA treatment did not show a significant result. TS significantly dropped in both children and adults, based on the copro-Ag ELISA, indicating that a single approach intervention of multiple rounds of MDA targeting a proportion of the population (SAC) can impact on transmission and spill-over into the pig population and the untreated adult human population. The second article by Braae et al. (2017) could not show a spill-over effect in the human population, but the author believes that this would become clear after a longer period and when having a bigger adult sample population. Both articles of Braae et al. showed a significant effect of a single human treatment strategy based on repeated MDA rounds on the prevalence of TS/PCC. In both studies only a sub-population, the SAC, were targeted, which might be an interesting strategy as it is more feasible and cost-efficient compared to community based MDA. However, the contribution of additional track and treat of TS cases in the whole population to the decrease in prevalence cannot be calculated, questioning the efficacy of multiple rounds of MDA treatment to SAC without the additional track and treat. Co-endemicy of schistosomiasis and T. solium (Braae et

al., 2015a) and increased availability of donated PZQ for the treatment of schistosomiasis support an integrated approach for both parasites and the article by Braae et al. (2016b) confirms that this strategy might work. However, the higher dose recommended for the treatment of schistosomiasis, might increase the risk of epileptic seizures in HCC persons (see higher), which is a major concern and has to be evaluated. A follow-up period after the last MDA round was absent in both studies, so the prolonging effect of the interventions remains unknown. Based on the MM by Kyvsgaard et al. (2007), the prevalence will return to pre-treatment levels after the intervention. As only a part of the population (SAC, 34% of the total population) is targeted and not all of these children are present or give consent for treatment,

27

transmission will still continue. Moreover, when treating based on the national schistosomiasis control program, the total MDA rounds would depend on its prevalence. In the article of Braae et al. (2015a), co-endemicy of T. solium and schistosomiasis was confirmed on country level, but co-distribution-maps on district or even on village level are still sparse due to a lack of data. Identification of co-endemic clusters on the same administrative level as MDA is carried out is important for multiple reasons. Firstly, significant variation in disease distribution on smaller scale is expected for both parasites as transmission depends on the presences of the respective intermediate hosts. In some populations, the prevalence of schistosomiasis might be low and the prevalence of T. solium high, leading to fewer MDA rounds, not sufficient for T. solium control. Secondly, this knowledge is important to know which areas are at risk for the side effects of PZQ. As a consequence, careful assessment of the populations to be targeted in combination with the implementation of additional intervention tools such as HE and porcine treatment - a One Health approach - is needed to reach sustained control and eventually elimination. In the study of Ash et al. (2015), the effect of two MDA rounds of a triple albendazole treatment, undertaken five months apart, on the prevalence of TS and STHs was measured. Albendazole was the drug of choice because of its broad spectrum efficacy against both T. solium and STHs

(Horton, 2000; Steinmann et al., 2011), absence of severe adverse effects (as opposed to PZQ) and acceptability to the government. A significant reduction in levels of both TS and STH prevalence was achieved by a triple dose albendazole treatment regime, but the increased STH prevalence, detected between MDAs, reflects the problem of reinfection through inadequate behavior (open defecation) and lack of sanitary conditions. Additional measures which support behavioral changes (such as HE), improved sanitation, and sustained chemotherapy programs, which may also need to tackle reservoir hosts, may therefore be needed to prevent transmission and obtain sustained results. Also here, the conclusion can be made that including T. solium control in broader STH and/or sanitation programs can be an efficient strategy. Single MDA often has temporary effects if the risk factors remain unchanged. This was modelled by Kyvsgaard et al. (2007) and confirmed by several articles previously discussed (Braae et al., 2016a; Johansen et al., 2017; Winskill et al., 2017). Moreover, a lot of non-infected people are treated which implies spilling of resources and associated risks. For these reasons, it is important to look at the effectivity and cost-effectiveness of alternative approaches as selective treatment and focus based treatment. Selective treatment of TS cases as a stand-alone strategy remains difficult, considering the difficult identification of TS cases by their vague symptoms and the unavailability of sensitive, specific, field-friendly detection methods in endemic areas (see 1.3). In addition, selective treatment is more expensive than MDA based on the currently available diagnostics and treatment (Alexander et al., 2011). The epidemiological basis of removing carriers out of the population is however undeniable and a recent MM by Winskill et al. (2017) calculated an annual track and treat strategy to be very effective (see 2.4, A). Research on development of better diagnostic tools is currently ongoing (see 1.3) and if successful, feasibility, availability, sensitivity and costs might improve in the upcoming years. Focus based treatment based on medical or veterinary data was proposed earlier as another cost-effective strategy (Pawlowski, 2008), but till now, data on cost-effectivity are still lacking due to inadequate record-keeping and surveillance of new cases. Alternatively, focus based screening and treatment of human (and pigs), based on the presence of a heavily infected pig is another recently proposed strategy (O’Neal et al., 2014) and might increase participation levels from residents living in these high risk areas, reduce costs and become a sustainable, permanent program. A 100 m. radius was proposed to screen and treat people when a heavily infected PCC pig would be found, based on small Peruvian study that found an eight-time higher prevalence of TS in people living within 100 m. of heavily infected pigs (O’Neal et al., 2012). The efficacy of this approach was tested in a prospective pilot study by O'Neal et al., 2014. Every four months, examination of the tongues of all pigs for nodules characterizing PCC was performed. Afterwards all residents living within 100 m. of a positive

28

pigs were screened using copro-Ag ELISA. Positive TS residents with were treated with NCL. The ring-screening reduced the transmission of T. solium, based on the sero-incidence among

pigs born during the intervention period in this pilot study (41% reduction in sero-incidence in the intervention village compared to baseline (incidence rate ratio 0.59, 95% CI 0.41–0.87). Therefore, it may provide an effective and practical approach for regions where resources are limited. The lower prevalence of confirmed TS in the intervention community at study completion compared to the control community (nearly four times lower, 95% CI 0.08–0.91), provides additional supporting evidence that ring-screening was effective. However, the difference of this secondary outcome may be statistically significant, the study was not powered enough to measure it. In addition, considerable variation in sero-incidence in the intervention village was measured, the study was not randomized, only two villages were included and PCC infected pigs might have been overlooked, as tongue palpation is often only positive when the pigs are heavily infected. These limitations suggest that testing this strategy in larger studies with longer follow-up time is necessary to validate the approach and determine the long-term effects. Moreover, the benefit of antigen detection over tongue palpation should be assessed for the detection of infected pigs in this strategy. Later, the study by Pray et al. (2016) concluded, in accordance to a study performed in Western Kenya (Thomas et al., 2013) that the estimated 100 m. radius is an accurate representation of pig roaming range in Latin America. Pigs were measured to roam a median of 82.8% of their time within 100 m. of their home. Furthermore 93% of time interacting with human defecation occurs within 100 m of pig residences, making it the area with the majority of exposure to human feces. Younger pigs were found to have significantly smaller home ranges, causing them to spent more time interacting with open defecation areas than any other age category. The latter suggests that young pigs are more likely to be exposed to human feces, and may be at a higher risk of infection from T. solium eggs than older pigs. The spatial relationship

between tapeworm carriers and PCC pigs was confirmed by Pray et al. (2017), who concluded that PCC prevalence was greatest within 50 m. of TS cases but that heavy cyst burdens were not necessarily to be found closer to TS cases. Articles by Pray et al. (2016, 2017) and Thomas et al. (2013) support interventions targeting the areas immediately surrounding heavily-infected pigs in two different areas, where T. solium transmission is most likely to occur. Studies

targeting both human and pigs within 100 m. ring area were conducted recently and results are eagerly awaited.

29

C. Health Education Table 4. Summary on the recent field trials involving health education. (av: average, BL: baseline, Child.: children, CI: confidence interval, ctl.: control, HCC: human cysticercosis, int.: intervention, m.: month(s), PCC: porcine cysticercosis, pi.:pig(s), TS: taeniasis, w.: weeks, X: no results for this variable, y.: years, ≠: difference)

Country, region

Year Population size/coverage

Intervention Improvement Follow-up period

Random Control Citation

Knowledge Practice/attitude Active HCC

+ -

Tanzania, Mbulu

2010-2012

2700 child., 60 schools

Video show, education by trained teacher, leaflet.

Increased 10% (int.) and 6% (control) after 6m. on TS, PCC, HCC, epilepsy.

Condemning infected meat (int.), how best to raise pi. (control).

Contacting a veterinarian for infected pi.

X Immediately, 6m., 12m.

Yes Yes (Mwidunda et al., 2015)

Tanzania, Mbeya

2014 79 professionals

Presentation, work on computer with ‘The vicious worm’.

Improved, immediately (p=0.001) and 2w. after (p<0.001).

efficient, simple, appealing.

computer-based design, suggestion: supplement leaflets

X Immediately, 2w.

No No, BL used as control.

(Ertel et al., 2017)

Zambia, Katete District

Jul’ and Nov’ 16

3 primary schools, 99 students

1/2-day workshops using ‘The vicious worm’.

High at BL (av. 62%), significantly improved immediately after (p<0.05 (part of the questions in 1 school), (p<0.001 for all questions in 2 schools).

X X Planned in study neighborhood.

No No, BL used as control.

(Hobbs et al., 2018)

Burkina Faso,

Boulkiemdé, Sanguié and Nayala

2011-

14

60 villages (2

excluded), 4645 eligible pp.,

Screening and discussion

of a movie, and a Self-esteem, Associative strengths, Resourcefulness, Action planning, Responsibility (SARAR) approach via the Participatory Hygiene and Sanitation Transformation (PHAST) model.

X Increase of

proportion HHs with latrine.

No difference

in pi. penning

Effective in 2/ 3

provinces. Decrease in cumulative incidence: ratio=0·65, (95% CI 0·39–1·05) and decrease in prev. proportion ratio=0·84 (95% CI 0·59–1·18). from BL to after int.

BL,18 m.

(before intervention), 36 m (18 m. after int.).

Cluster-

randomization

Yes (Carabin et

al., 2018)

30

The Landscape Analysis (Thomas, 2015) stated that HE, as many other measures, as a stand-alone strategy is not sufficient in terms of efficacy and cost-benefit to control T. solium. Though, it is proven that the prevalence of T. solium cysticercosis and TS can be reduced by changing attitudes and practices (Sarti et al., 2003; Ngowi et al., 2009) and furthermore it was shown that HE based on sound theoretical frameworks is most likely to be effective (WHO-EMRO, 2016). Thus, HE was recommended in addition to other strategies and for long-term control of the parasite. Since 2013, six articles related to HE were published. Four on field trials (Table 4.) and two on non-field trials.

In 2014, ‘The Vicious Worm’ was introduced as a computer-based T. solium education tool (Johansen et al., 2014). The suggestion was made to include the tool as an evidence-based specific control measure in any control program, next to vaccination and drugs. From its introduction on, the computer-based education program has been assessed for its efficacy in knowledge uptake, cultural acceptability and compliance in different test groups and information of these assessments has been used to adjust and update the tool. ‘The Vicious Worm’ fulfils some gaps in existing HE. Firstly, in most of the control programs, HE is an additional non-specific measure integrated with other primary strategies, such as medication or vaccination. Therefore, HE is often not properly assessed for its efficacy and impact. ‘The Vicious Worm’, aims to be a specific measure, supported by evidence on its efficacy in knowledge uptake, cultural acceptability and compliance. Secondly, a computer-based tool was found to be the optimal approach for sharing and assessing the best practices globally nowadays, including distribution of HE messages to relevant stakeholders and local communities. Other advantages include the reduction of teacher training costs, provision of uniform education and possibility to continuously improve the computer-based program. At last, ‘The Vicious Worm’ wants to bring a simple and meaningful message, tackling various difficulties (reaching stakeholders across disciplines and sectors, clarifying the complicated lifecycle,…) and misunderstandings (the association of the disease with superstation and witchcraft or the belief that you can’t get the disease if you don’t eat pork,…). A first assessment of ‘The Vicious Worm’ education tool on knowledge uptake and attitude towards the program among professionals in the endemic Mbeya area in Tanzania was published by Ertel et al. (2017). Professionals were chosen as study subjects as they play a key role in passing on information to high risk populations in rural areas. A presentation on the purpose of the study and the HE program and the prevalence and burden of T. solium TS and

cysticercosis in the local area was given, followed by individual or group work on a computer with the program. The study included 79 study subjects, both of the health and agricultural sector. Knowledge uptake was assessed at baseline, immediately after and two weeks after the intervention based on questionnaire surveys. The study subject’s overall knowledge was significantly improved both immediately after (77% (95%CI:67.7-86.3), (p<0.001)) and two weeks after (70%(95%CI:59.7-80.3), (p<0.001)). The knowledge regarding specific aspects too, except for acquisition and transmission of T. solium infections and the relation between

PCC, HCC and TS. Positive attitudes towards the program were found after focus group discussions, only the computer-based design was mentioned as a limitation for some study subjects and leaflets as supplement for rural areas were suggested. In contrast with the articles by Maridadi et al. (2011) and Mwidunda et al. (2015), neither gender nor educational level, were shown to be significant factors for knowledge uptake. These findings suggest that ‘The Vicious Worm’ is able to improve knowledge, regardless of gender, level of education or experience with T. solium and computers. Employment in the health sector compared to the

agriculture sector was found to be a significant factor for improvement of knowledge in both post-intervention studies. An assessment of the knowledge uptake among primary school students was published by Hobbs et al. (2018). Half-day workshops including a brief introductory session, a fun practice quiz, a ‘pre’ questionnaire, ‘The Vicious Worm’ educational component, comprising the introductory and village program sections and a ‘post’ questionnaire were organized in three

31

primary schools in Katete district. Pre- and post-intervention multiple-choice questionnaires found an improved knowledge score in 99 students immediately after the workshop. Also key messages to prevent transmission were better understood. Details on the parasites lifecycle were not fully understood, which was also seen in the publication of Ertel et al. (2015). This finding reflects the complexity of the parasite’s life cycle and the need to simplify concepts. Also for this target public, ‘The Vicious Worm’ was found to be an effective tool for the short-term T. solium education. Follow-up studies were mentioned to be planned to assess the long-

term impact of the program on knowledge uptake in the study neighborhoods. Furthermore, studies on behavioral changes and the effect of ‘The Vicious worm’ on the reduction in disease burden should be conducted in the future. Another attempt of creating a science-based HE intervention strategy was made by Ngowi et al. (2017). An optimal strategy for the endemic region of Burkina Faso was singled out using the PRECEDE-PROCEED (Predisposing, Reinforcing, and Enabling Constructs in Educational Diagnosis and Evaluation) model. The model is a planning model based on the idea that an educational diagnosis precedes an intervention plan and enhances the potential appropriateness and effectiveness of interventions. It includes a social and epidemiological assessment, a behavioral and environmental assessment, an educational and organizational assessment and an administrative and policy assessment. This model included group discussions to understand the community perceptions related to TS and PCC and rendered transmission risk factors (social assessment of PRECEDE). Also a close ended questionnaire was used during the study to quantify knowledge and practices related to T. solium

transmission (behavioral and environmental assessment phase of PRECEDE). In the end, the model led to an understanding of the needs, strengths and weaknesses in the control of T. solium infections specific for this region. The main problems identified in Burkina Faso area

were the lack of knowledge on TS and cysticercosis, and problems maintaining hygiene and sanitation in combination with the practice of free-roaming animals. The practice of free-roaming pig husbandry is an economically rational strategy for an impoverished population, which is unlikely to change. Though, the main issue concerning the transmission of T. solium

cysticercosis is the availability of contaminated faeces for consumption by these animals. If the environment can be made free from human faeces, most of the other identified risk behaviors (such as drinking unboiled water, letting pigs roam free, and eating partially cooked pork) would have a low impact in the transmission of the parasite. During the assessment, no reasons for significant knowledge differences between provinces in Burkina Faso could be found, emphasizing the need to implement HE interventions on a specific base for different regions. Based on the PRECEDE model, a multicomponent educational intervention approach to tackle the identified issues was developed. This intervention included a SARAR (Associative strengths, Resourcefulness, Action planning, and Responsibility) and PHAST (Participatory Hygiene and Sanitation Transformation) approach model (WHO, 1997), including community based training sessions on building latrines, latrine use and open defecation. In this way the program aimed to improve the communities’ self-efficacy in the implementation of cysticercosis control measures. In addition, PHAST model was supplemented by a 52 min. film, a discussion summarizing and identifying the key messages of the film and a comic booklet to improve knowledge about T. solium transmission and the benefits of its’ control. Results of this first

cluster-randomized, community-based, drug free study in SSA on the cumulative incidence and prevalence of active HCC, showed a decrease in both outcome measures and the efficacy of the intervention in two of the three provinces included in the study (Carabin et al., 2018). The differences in ethnic groups and social structures might have had a role in the non-efficacy found in one province. In addition to another PRECEDE study in Tanzania (Ngowi et al., 2008), that measured a 43% PCC decrease after education, but that did not seek to increase community self-efficacy, an increased proportion of HHs was measured to have a latrine. Overall, this study shows that community-engaged participatory interventions have the potential to be a low-cost control intervention strategy for cysticercosis in some low-resource settings. The low cost nature enables implementation at a larger scale, but research based

32

area depending adaptations might be necessary for ensuring the effectiveness of this intervention. When implementing a HE strategy, it is also important to consider the potential of the target population in terms of knowledge uptake and change of attitudes. HE was found to be useful in controlling TS and cysticercosis as mentioned above, but trials so far, have mostly been targeting adults. Alexander et al. (2012) reported improved knowledge and practices after a HE session to school children in India, but the lack of a control group made the study unable to estimate the actual effect of the intervention. Elsewhere, school children have been found to be good targets for messages to control other health problems and also their quality as good knowledge carriers has been highlighted (Lansdown et al., 2002). The recent article by Mwidunda et al. (2015) could demonstrate the potential of HE to school children for the control of T. solium cysticercosis and TS in endemic areas for the first time. A knowledge improvement of 10% after six months and a positive effect regarding practices towards the disease were found. A simultaneous improvement of 6% in the control group was contributed to the administration of the questionnaire twice in a short interval, which might have stimulated interest in the topic and led the children to think through their answers more carefully. Also differences between primary and secondary school pupils were found regarding knowledge and practices. The suggestion was made that a higher exchange of information exist among secondary school pupils, as they interact among themselves and across schools in contrast to children of primary schools, who use most of their free time playing games. Also at home, primary school pupils are the most directed by their parents to perform some family duties. Secondary, older school pupils are more respected, which may give them more time to meet with their peers for socialization. The study also found that increased age was associated with positive attitude to condemn infected pork and consult with a veterinarian for infected pigs as well as better scores for the knowledge part. This is most likely because of generally increased understanding on the health risks as the age increases. Future studies might assess the length of time to which the acquired knowledge would persist as well as its contribution to behavior change and reduction in disease burden.

D. Improving Pig Husbandry

D1. Vaccination

Human and pig T. solium infections seem to be susceptible to immunological interventions as it is common to find spontaneously destroyed cysticerci, surrounded by several immune cell types in activated states. These cysticerci also express molecules with immune activity (Sciutto et al., 2015). Additional evidence suggests the existence of acquired immunity and a temporary protection induced by primo-infection (Aluja et al., 1999). In the past, a number of vaccines have been created and tested under experimental conditions, based on related cestode antigens, of synthetic origins or by recombinant technologies. These candidates were also tested by DNA vaccination. The first report of effective vaccination against PCC was in Mexico.

Vaccine

Type

Population

Protocol Protection (anti-TSOL18-specific IgG titers)

Follow-up

period

Random

Control

Citation

TSOL18

Experi

mental

50 Landrac

e-Pietran pi. (12

wo., half ♂, half ♀.)

2 doses, im.

(neck), 4,8,12,16 or 20 w.

apart.

- 100% response - No diminution in ab responses for

doses up to 20 w. apart. - Titers in groups receiving the 2nd immunization more than 4 m. apart

developed higher mean ab titers than pigs receiving their 2 doses 4 w. apart.

- Animals immunized at an interval of 12 w. had 3/1 times the ab titer of those immunized at an interval of 4 w.

X Yes Yes (Lightowlers et al.., 2016)

Table 5. Summary of one field trial on pig vaccination. (ab: antibody, im: intramuscular, m.: months, pi.: pigs, w.: weeks, wo.: weeks old, X: no information for this variable, ♂: male, ♀: female)

33

The vaccine was based on a total extract of cysticercal antigens from T. solium cysticerci, drawn out from naturally infected pigs was in Mexico. Later, the three peptides responsible for protection were isolated and synthetized to produce the SP3Vac. The offset of another vaccine is based on the discovery that 18- and 45-kDa recombinant antigens from T. ovis give high protection against PCC. Corresponding T. solium antigens were then isolated as TSOL18 and TSOL45-1A and were found to be more efficient to protect against experimental cysticercosis infection in pigs. To this date, SP3Vac and TSOL18 have progressed the furthest and have been tested under experimental and field conditions (Aluja et al., 1999). As restricting pig husbandry to pens is currently not seen as a possibility by farmers (Thys et al., 2016; see 2.4, D3), only two measures remain to break the parasite life cycle on the pig’s level, being OXF treatment and vaccination. An issue to vaccination remains to address the influx of new born susceptible piglets into the population, as pigs in rural areas do not have a breeding season. Several strategies have been suggested to counter this problem: vaccination of young age piglets, which is not practical and ineffective due to presence of maternal antibodies and vaccination of pregnant sows which might endanger the offspring. An alternative strategy, treatment of pigs (OXF) combined with vaccination (TSOL18), was proven efficient in the study by Assana et al. (2010). The TSOL18 (Cysvax®) vaccine was reported to be registered and available for sale upon November 2016 in India and OXF 10% (Paranthic®), was registered in Morocco in 2013 and is currently the only registered drug for PCC treatment. Registration processes for both the anthelmintic and the vaccine are currently underway in other endemic areas3. The use of this strategy has previously been recommended as a short-term vertical control program followed by a long-term sustainable horizontal program with the potential for eradication in two seasons (Assana et al., 2013). Though effective (99% efficacy in protecting pigs against infection when used in conjunction with a single dose of OFZ (Gauci et al., 2013)), the application of both vaccination and chemotherapy in pigs should be applied in a way that would be effective, feasible and sustainable under field conditions, taking local pig management practices into account. Lightowlers (2013) identified a schedule involving four-monthly treatment of pigs with both TSOL18 and OXF as being likely to achieve a high level of disease control, whilst minimizing the number of interventions that would be required on annual basis. To test whether a long interval between the first and second immunization, as applied in the suggested schedule results in sufficient protection, an assessment of the specific antibody responses in pigs immunized with the TSOL18 vaccine was made by Lightowlers et al. (2016a) by altering intervals between the two immunizations (4,8,12,16 or 20 w.) (Table 5.). Results of this trial show antibody responses up to intervals of 20 weeks and

suggest that immunizations with TSOL18 that are given at approximately three-monthly intervals provide continuing protection after the second injection. This was calculated based on the diminution of antibodies below 700 (the lowest peak anti-TSOL18 antibody titer recorded by Kyngdon et al. (2006)). Though, further field trials including PCC and TS measurement are needed to confirm the efficacy of a three- or four-monthly based TSOL18 vaccination/ OXF treatment schedule as suggested by Lightowlers (2013). Antibody titers have been previously used to evaluate responses to the vaccine that are associated with protection (Kyngdon et al. 2006; Assana et al. 2010), but a true efficacy claim requires PCC and TS outcomes to prove a reduction of transmission on the pig level and human level. In the end, these are the goals to target for diminishing the economical and health burden of T. solium. Susceptible non-treated or vaccinated pigs carrying cysts might also be imported from nearby village and consumed before treatment or vaccination. If the application of any vaccination and/ or treatment schedule does not result in a satisfactory reduction of transmission, record-keeping and treatment of these Trojan pigs might be a solution. If an effectivity claim can be made, application of vaccination could be addressed in a more flexible way, as antibody responses develop at least for intervals up to 20 weeks between the first and second immunization. Moreover, a combined OXF/TSOL18 vaccination approach addresses the gap in protection of susceptible newborn pigs in the population as early infected pigs are first treated for PCC with OXF and afterwards prevented from re-infection through vaccination. A four-monthly vaccination (TSOL18) and OXF treatment scheme, integrated in a short-term elimination

34

strategy together with HE and Human MDA was recently tested in a large-scale field study in Zambia. Preliminary results can be found further in this thesis (see 3.3).

D2. Pig treatment Table 6. Summary of one field trial on pig treatment. (nr.: number, OXF: oxfendazole, PCC: porcine cysticercosis, pi.: pigs, po: per os, TCBZ: triclabendazole, tx.: treatment, w.: weeks, <: smaller than)

Several studies on the efficacy of anthelmintic treatments against PCC have been performed in the past (i.e. albendazole, flubendazole, fenbendazole, nitazoxanide, and PZQ) (Mkupasi et al., 2013a). Almost all tested anthelmintic drugs are active against adult stages of cestodes, nematodes and trematodes, but the challenge has been to find drugs effective against the larval stage (Gonzalez et al., 2012). Among efficacious larval stage drugs are OXF, albendazole and PZQ (Gonzalez et al., 2012; Flisser et al., 1990). OXF (30 mg/kg) remains the drug of choice, due to the requirement of multiple doses for albendazole and PZQ, which is impractical in the field (Mkupasi et al., 2013a) and the reports of adverse effects as anorexia, lethargy, prostration and death (Gonzalez et al., 2012). A shortcoming to OXF is the lack of 100% efficacy against cerebral cysts (Gonzalez et al., 1998, Sikasunge et al., 2008), although it is thought that consumption of pork brain in endemic areas is not common in most endemic areas (Gonzalez et al., 1998). The widely used triclabendazole (TCBZ), another member of the benzimidazole family, next to OXF and albendazole, is efficacious against adults and larval parasite stages such as Faciola, Paragonimus, some nematodes and cestodes (Coles et al., 1986; Keiser et al., 2005; Richter et al., 2013). On these bases, Vargas-Calla et al. (2016) recently evaluated the efficacy of TCBZ against PCC (Table 6.). TCBZ in a single dose of 30 mg/kg was not efficacious against T. solium PCC in the study as there were no apparent

differences in cyst burden or appearance between the TCBZ treated group of animals and the control group on necropsy. Subsequent microscopic evaluation however, showed mild inflammatory reactions, which might indicate efficacy of a higher dosage or multiple dose administration. Multiple doses of TCBZ were previously reported to be efficacious against Echiniococcus multilocularis metacestode cystic larval stages in an in vitro study by Richter et al. (2013), but would be impractical under field conditions.

Anthelmintic

Type Population

Cyst burden (limb

muscles, degenerated, calcified

and viable)

Cyst appearanc

e

Adverse

effects

Follow-up

period

Random

Control

Citation

TCBZ/OXF Experimental tx. of

naturally infected PCC pigs.

18 pi. (Huancay

o, Peruvian highlands)

, 6 tx. TCBZ 1 x 30mg/kg

po, 6 1 x 30 mg/kg OXF po, 6

1 x sugar water po (control

group).

- Control: 1658 (131 -

2575) cysts. - TCBZ: 1414 (511 -

4052) cysts - OFZ: 259 (38 - 1953)

cysts. - nr. of cysts: OXF

group<TCBZ group (P<0.001).

- TCBZ: mild to

moderate inflammatory response

around cysts - OXF: 100

% degenerated cysts,

severe ifnflammatory reaction.

None 17 w. post-

tx.

Yes Yes (Vargas-Calla

et al., 2016)

35

D3. Sanitation Table 7. Summary on the outcome of the Total Led Control Sanitation (CTLS) program by Bulaya et al. (2015). (AFS: African swine fever, BL: baseline int.: intervention, m.: month(s), PCC: porcine cysticercosis, pi.:pig(s), pos.: positive)

Improvement of basic sanitation, hygiene and HE has proven to be an effective strategy for parasitic and infectious diseases transmitted by faeces (Fleury et al., 2013). Thus, another way of interrupting the lifecycle of T. solium is by preventing pigs from eating infective human fecal

material. To this end, either people can be prevented from open defecation by improvement of the sanitation or pigs can be prevented from scavenging, for example by restricting them in pens. Community Led Total Sanitation (CLTS) is an innovative community-based sanitation program which aims to reduce open-air defecation trough the construction of latrine pits. It is assumed that CTLS will lead to the control of poor sanitation-related diseases, including HCC/PCC. In 2007, UNICEF piloted in conjunction with the Government of Zambia the CTLS approach in the southern province of Zambia, with a promising outcome: sanitation coverage increased from 23 to 88%, and 75% of the villages were open defecation free. Afterwards, ‘The 3 million People Sanitation Program’ was launched by the Minister of Local Government and Housing in Zambia. Twelve districts were included in a pilot study during including Katete in the Eastern Province. A preliminary evaluation of the effectiveness by Bulaya et al. (2015) (Table 7.) could

however not repeat these promising results. Eight months after the implementation of CTLS in nine villages of Katete district, sero-prevalences of PCC did not significantly improve and the knowledge, attitudes and practices did not change. Thus, introduction of latrines, did not guarantee the use of them, as it was observed that not all HHs had a latrine and the newly built latrines were not used. Moreover, there was a lot of variation between the villages. As a consequence, TS infected persons proceeded contaminating the environment by open defecation, which probably resulted in an insignificant decrease in infected pigs. Results of questionnaires suggest that a lot of cultural practices and traditional beliefs hamper the latrine usage. For example, open usage of latrines is considered taboo, while going into the bush is considered appropriate. Furthermore, occupants of a HH will not use the same latrine in relationships such as adults with children or in laws with parents. It is recommended that the

Co

un

try/

reg

ion

Year

Po

pu

latio

n

Inte

rve

ntio

n

Improvement Kn

ow

led

ge

Fo

llow

-up

Ran

do

m

Co

ntro

l

PCC (serum-

Ag ELISA)

Sanitation practices and attitudes

Zambi

a, Katete Distric

t.

Apr.

- Jun. ’12.

65,865 pi.

48,417 pp. 104 pi. (pre-int.),

275 pi. (post-int.).

64 (pre-int., 19% response

rate) and 89 respondent

s (post-int. 26% response

rate)

CLTS:

construction of pit

latrines.

BL : 14

pos. (13.5%)

Post-int.: 45 (16.4%)

pos. (p: 0.473).

- Crop season: pi. are kept

more in pens. - More toilets: 43 (67.2%), pre- and 74 (83.1%) post.

intervention. - Increase of latrines presence: pre (43, 67.2%)

- and post-int. (74, 83.1%) (p=0.027). -Latrine usage: no

increase: 41 (93.2%) at BL and 62(84.9%) post-int. giving a net increase of

only 21 latrines (p=0.15). This means that there has only been a 33.9%

increase in toilet usage (p=0.15). - Home slaughter:

common practice - No change in selling pork with cysts pre- and post-

int. (p=0.679) - AFS: most important pig disease (p=0.00)

- 80%

had heard about/

observed PCC.

8 m. No,

villages were chosen on

certain criteria.

Yes

36

installation of latrines, which is still ongoing in Katete (because of the lack of material), is combined with HE and sanitation programs that do not only address the health benefits, but also focus on the local context and the sanitary-related taboos, to obtain also an increased usage of the latrines. These findings were also recorded by Thys et al. (2015), who assessed the communities’ perceptions, practices and knowledge regarding latrines through 21 focus group discussions in separate male and female groups within seven villages in the Petauke district (Eastern Province, Zambia). The study revealed the same sanitary taboos and found that latrines were not constructed in every HH because of the convenient use of existing latrines in the neighborhood. Moreover, Thys et al. (2015) found that mostly men are responsible for building latrines and mostly men prefer open defecation. Therefore, the suggestion was made to address sanitation programs including sanitary related sanitary taboos to men in order to obtain latrine usage and abandon open defecation. Thys et al. (2016) also identified possible barriers to pig confinement by a total of 21 focus group discussions on pig husbandry practices in seven villages from Petauke district (Eastern Province, Zambia). The study revealed that pig confinement is currently not seen as an acceptable method to control PCC by farmers in Eastern Zambia, based on the pigs’ role in society (financial, agricultural and traditional), the distribution of the management tasks among the family members owning pigs (feeding, building kraal, seeking care) and environmental aspects (feed supply, presence of bush, wood use priorities, rainy season). Even if negative aspects/health risks of free-range pig keeping are perceived, people are ready to take the risk for socio-economic reasons. Finally, here again, gender plays an important role because women, and also children, seem to have a higher perception of the risks but lack power in terms of economic decision-making compared to men. Addressing men concerning this matter during HE sessions might be a good strategy. If pigs were to be held in pens as a complementary control strategy, next to treatment/vaccination, farmers would probably be most motivated to hold pigs in pens during the crop season, to prevent the crops from being eaten (Bulaya et al., 2015). Nevertheless, in a sero-prevalence study Braae et al. (2014b) remarked that confined pigs did not have a lower PCC sero-prevalence compared to free-roaming pigs. Elevated pens, pens with a dirty floor, open latrines and feeding potato peels were identified as PCC infection risk factors in a follow-up case-control study (Braae et al., 2015b). Improving pig management should therefore not be limited to confinement, but should also adress these risk factors. Furthermore, the contribution of the environment on the transmission of T. solium and the

survival of eggs remains to be determined.

E. Combinations Table 8. Field trials on combination strategies. (CI: confidence interval, cov.: coverage, d.:day(s), HH: household, m.: month(s), MDA: mass drug administration, NCZ: niclosamide, OXF: oxfendazole, pi.: pig(s), pp.: people, po: per os, prev.: prevalence, tx.: treatment, vacc.: vaccination, y.: year)

Countr

y, region

Year Population

size and coverage

Intervention Outcome Follow-

up period

Ran

dom

Cont

rolled

Citatio

n

Peru, Tumbes

2007?-?

107 villages, 81,170 pp.

(84.7% cov.), 55,638 pi.

Human MDA (NCZ, 3 rounds) & pi. MDA and vacc.(OXF, every 2m. +

TSOL18, 2 rounds of 2 vacc.).

3/342 pi.: live, non-degenerated cysts, no infected pi. in

105/107 villages. 1 y. later: 7/310 pi.: live non-degenerated

cysts, no infected pi. in 11/17 villages.

1m., 1 y. No No (Garcia et al., 2016)

Northern Lao, Mai

District

Oct. ‘13 – Jan.

‘15

300 pp. (55 HHs), 63% (MDA 1)

and 65% (MDA 2) cov. 414 pi.

(90% cov.).

2 rounds of 3d. human MDA (albendazole 400 mg, 10/13 and 03/14.), 3

rounds of TSOL18 vacc. & po pi. tx. (OXF, 30 mg/kg, 10/13, 03/14 and

10/14), repetition after 1 m.

78.7% decrease in TS population prev. From 30.6% (95% CI: 25.5-

38.9%) to 6.5% (95% CI: 3.4-9.5%). Significant reduction.

12 m. No No (Okello et al., 2017)

37

As T. solium is a zoonotic disease, affecting both pig and human health and its’ transmission

is influenced by several factors such as sanitary facilities, pig husbandry, social and cultural behavior and beliefs, many options to target the parasite are available. However, MMs (Kyvsgaard et al., 2007; Braae et al., 2016a; Johansen et al., 2017; Winskill et al., 2017) suggest that both pig and human treatment are required to get a quick and sustainable impact. Up till now, three strategies have been attempted or modelled, including the combination of pig MDA/vaccination and human MDA (Garcia et al., 2006; Garcia et al., 2016; Braae et al., 2016a; Okello et al., 2017; Johansen et al. 2017; Winskill et al., 2017), the combination of pig vaccination and human HE (De Aluja et al., 2012) and the combination of pig vaccination and human MDA (only modelled by Kyvsgaard et al. (2007)). Adding porcine vaccination to the human and porcine MDA combination strategy has been recommended as a control strategy by the Cysticercosis Working Group for East and Southern Africa (CGWESA, 2011). In this review, two field trials on the combination of human and pig MDA combined with pig vaccination are included, one measuring the effect on TS prevalence and the other on elimination of transmission (Table 8.).

Okello et al. (2017) was the second (after Garcia et al. (2006)) to find that a combination of both human and porcine MDA can result in a significant decrease in human TS levels in a relatively short period of time. The study was the first to test this in a Southeast Asian context and provided the first data on the impact upon the adult parasite in the human host. A significant (p<0.0001) reduction was measured as TS prevalence, diagnosed trough copro-Ag ELISA, decreased with 78.7%. Prevalence dropped from 30.6% (95% CI: 25.5–38.9%) before the first intervention to 6.5% (95% CI: 3.4–9.5%) after the last intervention. However, this study could not measure short-term impacts on PCC or HCC as the pigs could not be dissected and no human blood samples could be provided due to cultural beliefs and practices. Furthermore, there is a lack of alternative specific diagnostic assays that do not require blood in Asia. Though, as cases of NCC are suspected to be present (Okello et al., 2014), long-term assessments of the impact of a human-porcine approach on HCC is required. Also sufficient coverage remains an issue. In this study, the human coverage of 63-65% of the population was compensated by a coverage of 90% of the pig population. However, transmission by pig meat can even occur after implementation of this strategy through consumption of pigs before slaughter age (<6 m.) or pigs from outside regions. This was confirmed by three new cases of TS, who had at least received one full course of albendazole treatment. A similar approach was used by Garcia et al. (2016), with the aim of eliminating T. solium in

an endemic area of Northern Peru. This three-phase program, started off testing and comparing elimination strategies in terms of feasibility and effectivity of interrupting the transmission of T. solium infection in 42 villages. In a second phase the two most promising

strategies from intervention one, mass treatment, and mass screening and treatment (both with or without vaccination of pigs) were compared in 17 villages. In a third and final phase, the final strategy of human MDA combined with pig MDA and vaccination was implemented in the entire rural region of Tumbes (107 villages). The study achieved interruption of transmission in 105 of 107 villages through a one-year attack approach and elimination persisted in most areas for at least one year without further intervention. The one-year study-design was however not controlled and very intensive, including short intervals between rounds of MDA and adding a vaccine. Moreover, temporary effects are expected if additional activities are not implemented. These aspects raise the question whether the elimination goal is a practical and economically viable target. Other, controlled experiments with less-intensive and expensive strategies are needed to answer this question. The first results of a controlled two-year integrated elimination program in the CYSTISTOP project (Zambia), combining HE, human MDA, pig MDA and vaccination can be found further in the thesis (see 3.3). In this project, additional TS outcomes were measured by copro-Ag ELISA, giving an idea of the impact of the strategy on tapeworm carriers.

38

3. Field Study

3.1 CYSTISTOP project

Current information on the optimal combination for cost-effective prevention and control of T. solium in endemic situations is limited and large-scale studies including multiple strategies have

never been carried out in Zambia (Kyvsgaard et al., 2007; Gabriël et al., 2016). For this reason, a six year during project named ‘CYSTISTOP’ was set up, evaluating the cost-effectiveness and acceptability of elimination and control of T. solium in a high endemic region of

Zambia, Katete. The project runs from 2014 to 2020 and consists out of five work packages: a systematic review, an optimization of the disease transmission model, an interventional field study, a socio–economic evaluation and a follow-up, reporting and dissemination of the data. The interventional field study is conducted in three communities in the Katete and Sinda districts of the Eastern province of Zambia, an area reported to be endemic for T. solium in both pigs and

humans (see 1.1). The communities are divided in an Elimination study arm (Nyembe, 1,210 people, 8 villages, 520 pigs), a Control study arm (Chimvira, 1,470 people, 11 villages, 827 pigs) and a Negative Control study arm (Herode, 1,329 people, 7 villages, 591 pigs). In the Elimination study arm (E-arm), elimination was intended by integrated measures on a short term, targeting both pigs and human. In the Control study arm (C-arm), a single control option, targeting only pigs is implemented, targeting control of the parasite on a longer time scale, by interventions in pigs taking place every 12 months. In the Negative Control study arm (N-arm) no specific interventions, apart from HE sessions take place (Figure 4.) (Protocol CYSTISTOP, 2016).

Figure 4. Schematic representation of the interventional field study CYSTISTOP, containing the Elimination (E), Control (C) and Negative Control (N) study arm. (MDA: human mass drug administration with praziquantel (PZQ); Ed: human health education; VACC: porcine vaccination with TSOL18; OFZ: porcine oxfendazole treatment. HCC: human

39

cysticercosis detection. TS: taeniasis detection; PCC: porcine cysticercosis detection, EI#1-6: interventions in the Elimination study arm, CI#1-5: interventions in the Contorol study arm) During the Erasmus project, PE, FC1 and FN1 (red frame) took place. In March, CI#3 took place (Figure based on: Protocol CYSTISTOP, 2016).

One of the objectives of this master thesis (see 1.5) is to determine whether elimination or rapid reduction of infected pigs can be obtained under Zambian conditions (smallholder, free-ranging pigs kept in field conditions and exposed to natural infections of T. solium in Zambia) after short-term integrated measures targeting both humans and pigs, as implemented in the E-arm. This study took place during a ten-week master student-exchange between the Department of Veterinary Public Health and Food Safety (Ghent University, Belgium) and the Department of Clinical studies (UNZA, Zambia), when several operational activities of the CYSTISTOP project took place. In January 2018, a first two-week post-elimination (PE) survey intervention in the E-and N-arm took place, including the sampling of people and pigs and detailed dissection of pigs (post-mortem examination, PME). In March 2018, a second intervention was conducted in the C-arm, whereby all eligible pigs were treated and pig blood sampling was conducted. Results of this second intervention in the C-arm are not included in the master thesis.

3.2 Material and methods

3.2.1 Study area

Figure 5. Map showing the two trial sites.

Nyembe neighborhood (E-arm, 245 HHs) consists out of five villages and two farms: Chikutu, Kayela, Mtalalika, Safari, Zonde, Apton farm and Peters’ farm. Herode neighborhood (N-arm, 269 HHs) consists out of seven villages: Chakumba, Chimwayi Chamoto, Etala, Herode, Kapelepeta and Robert. Both areas are located 32 km apart from each other (Figure 5.).

Zambia

40

3.2.2 Study animals

Pigs were predominantly free-ranging during their life span and typically kept by small holder or subsistence farmers. They were typical of pigs that are commonly raised in the study sites. Pigs could be confined for part of the day or of the year but were not housed or confined for their entire life span (Figure 6.). More details of the pigs can be found in Table 9.

Inclusion criteria

- Owner willing to allow pig to participate in all aspects of the study, including undergoing blood sampling and administration of an oral anthelmintic drench and vaccine

- Living within the study villages - Aged two months or older - Not late-term pregnant (in third trimester) or lactating sows - Owner willing to sell the infected pigs to the study team Exclusion criteria - Owner unwilling for pig to participate in some or all aspects of the study, including

undergoing blood sampling or administration of an oral anthelmintic drench - Living outside of the study villages - Younger than two months of age - Late-term pregnant (in third trimester) or lactating sows - Owner unwilling to sell the infected pigs to the study team

At each study site, animals that met the inclusion criteria were enrolled in the study provided that owner consent was given. An informed consent was signed to provide written consent for inclusion of the owners’ herd of pigs in the study for the whole study duration (until Augusts 2020). All animals in the study were owned.

In the study period, pigs enrolled but not selected for post-mortem examination (PME) (either at baseline or at the post-elimination survey) continued to be reared, reproduced and slaughtered as normal for that herd.

Table 9. Overview pig characteristics.

Species: Porcine (Sus scrofa domesticus)

Breed: Local Nsenga breed

Source: Owned by local farmers in Katete and Sinda districts

Body weight range: 5 – 110 kg at first intervention (03/2016)

Age: 3 – 36 months at first intervention (03/2016)

Gender: Male, female and neutered

Physiological status: None late gestational stage, healthy

Figure 6. Local eligible sow and non-eligible piglet at the trial site. Pigs were predominantly free-ranging. Some pigs were confined during the night in self-made pig houses, aiming to protect them from predators. The pig house can be seen on the right side of the image.

41

Identification: No identification tags before intervention. Two uniquely numbered ear tags (one in each

ear, both ear tags carried the same number) were placed

3.2.3 Human population

For the baseline survey, one randomly-selected consenting adult member and one randomly-selected assenting (and parental consent) child member (above five years) from each HH was sampled, whereas for the post-elimination survey in the E-arm, all eligible consenting members of the elimination communities were invited to participate. It is expected that all age, sex and socioeconomic groups were equally represented. No human sampling was conducted in the N- arm.

Inclusion criteria - Willing and able to participate in all aspects of the study, including providing blood and

stool samples, participating in a questionnaire survey and group discussions, and taking oral anthelmintic tablets (the latter specific for the E-arm)

- Willing and able to provide informed consent (signature or thumbprint with impartial witness; assent for minors with parental consent).

- Living in, attending school in, or regularly visiting the bore holes present in, the study communities

- Aged five years of age or older - People without epilepsy

Exclusion criteria

- Unwilling or unable to participate in some or all aspects of the study, including providing blood and stool samples, participating in a questionnaire survey and group discussions, or taking oral anthelmintic tablets (the latter specific for the E-arm)

- Unwilling or unable to provide written (signature or thumbprint with impartial witness) informed consent (or assent for minors)

- Living outside of, and not regularly visiting, or attending school in, the study communities - Children aged four years or younger - People with epilepsy (identified cases by the rural health center, questions included in

the registration procedure) - Seriously ill individuals (people unable to engage in the normal activities of daily living

without assistance because of their illnesses)

3.2.4 The elimination strategy

Measures were applied in the E-arm, during 24 months previous to the post-intervention study and included six four-monthly interventions (Figure 4.: EI#1-6) combining TSOL18 vaccination

(CYSVAX™), OXF pig treatment (30 mg/kg, Paranthic 10%™, MCI), PZQ human treatment (10 mg/kg) and HE. No measures, apart from HE were applied in the N-arm. TSOL18 was administered by intramuscular injection in the left side of the neck behind the base of the ear. OFZ was administered per os as a single dose of 3 mL/10 kg (30 mg/kg body weight) by graded dosing syringe. Pigs were weighed to determine body weight (live weight) using, a weigh scale. Human MDA was administered orally with a glass of water as a single dose of 10 mg/kg. Eligible candidates were weighed prior to administering an appropriate dose of PZQ. Tablets, provided free of charge by the WHO for use in this study, came in 600 mg formulation. HE included information regarding the T. solium parasite including its prevention, transmission,

clinical signs of infection, methods for disease control and prevention such as food-handling and

42

preparation methods, pig-management, hygiene, and sanitation. Education was delivered via village meetings/presentations, posters displayed at the rural health centers, leaflets distributed to HHs, and via the educational computer package ‘The Vicious Worm’5 , which was presented to SAC in the community schools. An overview of the key study dates can be found in Table 10.

Table 10. Overview of the key study dates.

3.2.5 Ethical considerations

This study was conducted within the ongoing CYSTISTOP project. Ethical clearance was obtained from UNZA Biomedical Research Ethics Committee (004-09-15) and the Ethical Committee of the University of Antwerp, Belgium (B300201628043, ECUZA16/8/73). The study was introduced and explained to all inhabitants and pig owners, both in village group settings and within individual HHs, prior to each field visit. Voluntary, written informed consent was obtained prior to enrolment in the interventional field study for each human participant or provided after assent by a parent or guardian if the participant was younger than 18 years of age. The informed consent form covered both participation in biomedical sampling and intervention rounds, including HE activities. Written informed consent was provided for treatment and vaccination and dissection of the pigs. Oral permission from all pig owners prior to the commencement of each pig sampling or intervention round was obtained. An incentive for participation was given every sampling or treatment round (soap or sweets for children). Pigs for dissections were purchased from willing pig farmers. Qualified medical health professionals performed all human MDA interventions and collected all human samples. All pig sampling, restraint, handling and treatments were carried out by professional veterinarians adhering to the Zambian regulations and guidelines on animal husbandry. All biomedical samples were assigned a study identification number so that laboratory technicians were not able to identify individual participants or the study arm they belonged to.

3.2.6 Post - elimination survey: Nyembe (E-arm) and Herode (N-arm)

In this survey the T. solium cyst prevalence after six intervention rounds (E-arm) was compared

to the prevalence in a similar population of pigs not given these interventions (N-arm) and to the baseline level by detailed full-carcass dissections and by detection of circulating antigen (serum). Secondary, the TS prevalence among people in the E-arm was measured by antigen detection in stool samples and compared to baseline levels. The serum and stool samples were analysed in the Regional Reference Laboratory for TS/cysticercosis in the Department of Clinical Studies, School of Veterinary Medicine, UNZA by the in house B158/B60 monoclonal antibody based Ag ELISA (serum) and the stool samples were analyzed by an in house copro-Ag ELISA. The identified (suspected) cysticerci were confirmed by molecular tools.

5 To be found on: http://www.theviciousworm.org/ (last consulted on 16 April 2018)

Description Date

Baseline post-mortem examination (BE, BN): 22-26/10/2015; 15-27/11/2015

First Treatment Administration (EI#1) 28-31/03/2016

Second Treatment Administration (EI#2) 20-23/07/2016

Third Treatment Administration ((EI#3) 22-25/11/2016

Fourth Treatment Administration ((EI#4):): 24-27/03/2017

Fifth Treatment Administration ((EI#5) 27-29/07/2017

Sixth Treatment Administration ((EI#6):): 28/11-02/12/2017

End post-mortem examination (PE, FN1): 22-27/01/2018; 14-16/02/2018

http://www.theviciousworm.org/

43

3.2.6.1 Sample size calculation

The number of animals for PME was calculated upon detection of an 80% reduction in PCC prevalence, with a power of 80% and with the assumption that an initial prevalence of 15 % to <1 % was present. Based on these calculations, dissection of 34 to 40 animals was required (Table 11.).

Blood samples were collected from all eligible animals (see 3.2.2) of consenting farmers in the E-arm and N-arm. All eligible (see 3.2.3), consenting members of the E-arm were invited to donate a stool sample. Table 11. Study Design and Summary of Study Pig Treatment Groups.

Study arm No. of Study

Sitesa

Pig

Treatments Regimen

No. of pigs

enrolledb

No of pigs for PME (baseline/post-

elimination)

Negative Control 1 None None not applicable 31/32

Elimination 1

TSOL18 (1 mL intramuscular)

and

OFZ (30 mg/kg per os)

Once every 4 months

(Days 0, 114, 239,

361, 486, 610 ±15 days)

Between 19 and 99 pigs per

intervention; in total

241 pigsc

37/26

a. A study site consists of one or more adjacent communities or villages.

b. Total number of pigs vaccinated/ treated.

c. Pigs were >8 weeks of age, at least 4 weeks from farrowing, and not clinically ill or intended for slaughter within 3 weeks of receiving OFZ.

Pigs for PME at baseline or during the post-elimination survey were selected randomly from the herds for which owners had given their consent to participate in the study. Preference was given to select as many different herds as possible to obtain a sample representing the whole village.

3.2.6.2 Preparation activities The evening before the start of the post-intervention survey, all team members received a training on the project (standard operating) procedures (SOPs), covering project related good clinical practices (GCP) and good clinical laboratory practices (GCLP). The project procedures included the euthanasia of pigs (Appendix 3.), the pig dissections (Appendix 2.), the pig blood sampling (Appendix 4.), the field lab work and the human stool sampling. Furthermore, sensitization was conducted in both intervention villages, Nyembe and Herode, to re-sensitize the villages for the stool samplings, pig sampling and dissections. Additionally, also HE was conducted, using large canvas poster, leaflets, pictures, plasticine models etc.

3.2.6.3 Human stool sampling and pre-treatment in the field lab (E-arm) Human stool samples were collected in the eight villages in the E-arm. Sampling was conducted at village level by three different teams, under supervision of a medical doctor. All HHs were visited, HH sheets were verified and updated (for example when new HH members or HH members moving out), informed consents were checked and updated (for example when a member turned 18) and stool pots were given to every eligible HH member (see 3.2.3). Stool pots were labeled with a pre-encoded barcode and the participants’ name. For children or illiterate participants, colorful stickers were used to individualize their stool pots. Participants could return stool pots the day after, when the teams returned to the visited HH to collect the stool samples or any time at the central spot in the study neighborhood or at the CYSTISTOP room overnight. Two

44

days after the stool pot distribution, door-to-door follow-up visits were organised, to collect more stool samples.

In the field lab, the overnight refrigerated stool samples were separated into two aliquots within 24 hours by dividing them into two 15 mL Falcon tubes labeled with the same pre-printed ID barcodes, an ‘F’ (formalin Falcon tube) or ‘E’ (ethanol Falcon tube) and the date. Subsequently, the aliquot in the ‘F’ pre-labelled Falcon 15 mL tube was covered and mixed with 10% formalin and the ‘E’ pre-labelled Falcon 15 mL tube was covered and mixed with 70% ethanol. If there was not enough stool material to make two samples, priority was given to the formalin sample. Samples were stored at room temperature and afterwards entered into EpiCollect and EpiData data management software packet.

3.2.6.4 Pig necropsys (E-arm and N-Arm) The evening before the dissection would take place, all eligible pigs of slaughter age were entered into an Excel spreadsheet, followed by randomization of the columns and selection of the first 37 pigs for dissection. The owners of these pigs were contacted to confirm the purchase. For each pig to be dissected, a “Pig Dissection sheet” was prepared with pig identification barcode stickers (“NYE-P-001” to “NYE-P-037” and “HER-P-001” to “HER-P-037”) and other details as can be found in Appendix 1. Pigs were then slaughtered by captive bolt and exsanguination as described in SOP Pig Euthanasia (Appendix 3.). During exsanguination, six tubes of blood (for serum preparation) were collected in 50 mL Falcon tubes, labeled with the appropriate pig barcode sticker and dated. Consequently, the carcass was prepared for dissection by skinning, removal of the organs and the head, cranio-caudal bisection into two equal halves and deboning. During this process, special attention was paid to the muscles lying just under the skin upon detection of cysts, to the cysts present in the eyes and the presence of T. hydatigena cysts. Initial dissection

of a half carcass, including organs and predilection sites was proceeded by 3 mm slicing and examination of all muscles, organs, body surfaces and cavities for cysts (T. solium or T. hydatigena or other). Predilection sites (masseter, heart, tongue, psoas, diaphragm, liver, lung,

brain, neck and oesophagus were always dissected in full. Other sites were dissected starting from half the carcass. If no cysts were found in the first half carcass, the other half was dissected too. Half carcass dissection or full carcass dissection was recorded on the Pig Dissection sheet. Viable, degenerated and calcified cyst counts per site, rendering into a total number of viable, degenerated and calcified cyst and a general total number was recorded on the Pig Dissection sheet. Also the number and location of suspected T. hydatigena cysts, the result of the tongue palpation (negative/positive) and other significant findings were recorded. Five or less muscular cysts, all cysts of other sites and doubtful cysts were collected and placed into separate 70% ethanol cryo tubes labeled with the pig barcode, the vial number, the date and “muscular” or the name of the site, and transported to Belgium for confirmation and speciation by PCR-RFLP as described by Dermauw et al. (2016). After completion of the dissection, all heavily infected parts of the carcass were collected into bin bags and immediately incinerated. Lightly infected carcasses with degenerated and calcified cysts, were released to the owner under the condition that they were boiled for at least one hour. Pigs were euthanized and dissected one by one and equipment and working surfaces were cleaned in between pigs to avoid contamination.

3.2.6.5 Pig blood sampling and pre-treatment in the field lab (E-arm and N-Arm)

Pig owners who previously gave informed consent to sample their pigs for the project were identified by the CYSTISTOP HH Master list and visited upon collection of blood samples. Before sampling, all pigs were entered in the Pig HH Registration Follow-up sampling sheet, including dissection pigs and non-eligible pigs. The previous Pig HH Registration sampling sheet was checked and updated (e.g. what happened to the animals that were previously present). If new pig owners were visited, a new informed consent form was drawn up. All eligible pigs (see 3.2.2)

45

in the HH were captured and pig information (age, sex, treatments and present ear tags) was recorded on the Pig HH Registration Follow-up sampling sheet. Consequently, pigs were sampled following the SOP Blood Sampling Pigs (Appendix 4, Figure 7.) and tongue palpation was conducted.

Successful or reasons for unsuccessful blood sampling attempts and results of the tongue palpation (positive/ negative/ reason for not tested) were mentioned on the Pig HH Registration Follow-up sampling sheet. Blood was collected in 10 mL blood tubes with a pre-printed barcode sticker, indicating the study area and the sample number. For Nyembe barcode labels “NYE-P-038” to “NYE-P-100” were used. For Herode, “HER-P-038” to “HER-P-100” were used. The first 37 labels “NYE-P-001” to “NYE-P-037” and “HER-P-001” to “HER-P-37” were used for the dissected pigs. Blood samples were stored overnight in the refrigerator (4°C) and processed in the field lab within 24 hours. The serum was separated from the blood clod by centrifugation of the samples

at 3000 rpm for 15 min. and pipetted into two pre-labelled and dated Cryovials tubes or 15 mL Falcon tubes (blood samples of dissected pigs). One Cryovial of each sample was stored in sample box A, the other in sample box B (back up) and stored in the freezer at -20°C. Afterwards, the blood samples were registered and uploaded in EpiCollect/EpiData.

3.2.6.6 Lab work

Serum samples Pig serum samples were processed following the Ag ELISA cysticercose protocol (ITM, 2015). In a first step, the samples were pretreated in order to break down immune complexes, to obtain free circulating antigen and moreover to reduce cross-reactions with sera of Trypnanosoma infected

individuals. To this end, 150 µL of the positive control serum sample or unknown serum sample was added to 150 µL 5% trichloroacetic acid solution (TCA) (2 wells per sample) and 75 μL of the negative control sera (1 well per sample) was added to 75 μL 5% TCA solution. Afterwards, samples were vortexed, incubated for 20 min., again vortexed and centrifuged for 9 min. at 12000g. Next, 150 (positive control and unknown serum samples) or 75 µL (negative control serum sample) of the supernatant was neutralized by adding it to 75 (negative control serum sample) or 150 µL (positive control and unknown serum samples) of neutralization buffer (pH 10). This resulted in a final 1 on 4 dilution of the samples.

During the pre-treatment of the samples, the ELISA plate (layout: Figure 8.) was prepared by coating the wells with 100 μL of capturing antibody (B158C11A10) (2.4 μg/mL coating buffer) except for the two substrate control wells, which were coated with 100 μL coating buffer (0.05M carbonate/bicarbonate buffer, pH 9.6). Afterwards, the plate was incubated for 30 min. at 37°C while shaking, followed by a one-time wash (phosphate buffered saline (PBS), 0.05%), blocking of all wells with 150 μL blocking buffer (PBS, 0.05% + 2% newborn calf serum (NBCS)) and again incubation for 15 min. at 37°C while shaking. The last preparation step for the plate consisted out of emptying the plate, without washing.

Figure 7. Blood sampling of an eligible pig during the post-intervention study following the SOP Blood Sampling Pigs (Appendix 4.).

46

Figure 8. ELISA plate layout: ?1 to ?40 represent the wells for the unknown samples, +1 and +2 (2 wells per sample) represent the positive controls (2 wells per sample) and -1 to -8 the negative samples (1 well per sample). SC and CC represent the conjugate and substrate control. Non-specific reactions between the plate, coating/blocking and conjugate are intercepted by the conjugate control. The quality of the substrate (e.g. by influence of light) can be traced by the substrate control. Both controls need to be negative (below cut-off value). Negative control samples (-1 to -8) are used to calculate the cut-off of the assay and should be matched to the species of the samples to be tested, to avoid any bias in the interpretation of the results. Positive control samples (+1 and +2) are used to see if the assay itself was successful.

Consequently, 100 μL of pretreated samples was pipetted in the designated wells of the ELISA plate. Wells for the substrate and conjugate controls were filled with 100 μL blocking buffer. The plate was then incubated for 15 min. while shaking and washed 5 times with washing buffer. Next, 100 μL of detecting antibody (B60H8A4) (1.25 μg/mL blocking buffer) was pipetted in all wells, except for the two substrate control wells, where 100 μL blocking buffer was added. Again, the plate was incubated for 15 min. at 37°C while shaking and washed 5 times with washing buffer. The, 100 μL of peroxidase labelled streptavidin (1/10000 in blocking buffer) was added to all wells, except for the two substrate control wells. In the latter, 100 μL blocking buffer was added. The plate was again incubated for 15 min. at 37°C while shaking and washed 5 times. While incubating, ortho phenylenediamine (OPD) was prepared in a dark recipient by dissolving one tablet in 10 mL of phosphate-citrate buffer. After incubating, 10 μL of H2O2, followed by 100 µL of OPD was added to all wells. Finally, the plates incubated for 15 min. at 30°C in the dark, whereafter, the reaction was stopped through adding 50 μl H2SO4 (4N) in each well. The plate was read at 492 nm. (max. absorption measure) and 655 nm. (background measure) by the Multiskan EX Microplate Photometer (Thermo Scientific). The intensity of the measured color, or the optical density (OD), reflects the amount of labeled antibody present in each well. After checking whether the two duplicate wells with positive and unknown samples roughly gave the same OD, the average OD was calculated for every sample. The cut off value was calculated based on the OD’s of the negative samples using a variation of the students t-test (Sokal and Rohlf, 1981). After calculation of the cut off value, ratios per sample were calculated as follows: ratio = average OD/cut off. When the ratio was >1, samples were considered PCC positive with a certainty of 99.9 %. A Microsoft Office Excel file was available to assist with these calculations.

Stool samples The formalin Falcon tubes containing human stool samples were processed following the copro-Ag ELISA protocol for the detection of Taenia tapeworm antigens in faeces (Mwape et al., 2012).

First, the stool samples were prepared by mixing equal volumes of PBS (0.05%) and faecal sample after removal of the overload of formalin. Afterwards they soaked for one hour, with intermediate shaking and were spun for 30 min. at 2000g. They rested one night in the fridge. One tablet of coating buffer was dissolved in 100 mL H2O (=0.05M carbonate/bicarbonate buffer, pH 9.6), after which IgG polyclonal (=1.39 mg/mL) was added to the coating buffer up to a concentration of 2.5 µg/mL. In the substrate control wells, only coating buffer was added. All other

47

wells were coated with 100 µL of the prepared solution. After incubation for one hour at 37°C while shaking, the plates were washed once (PBS, 0.05%), blocked (PBS, 0.05% + 2% NBCS) and then again incubated for one hour at 37°C while shaking. Consequently, 100 µL of the supernatant was added to the unknown sample wells and 100 µL of T. solium adult tapeworm antigen (1 mg/mL in blocking buffer) was added to substrate control and conjugate control wells. After one hour shaking at 37°C, the plate was washed 5 times (PBS, 0.05%). This was followed by adding 110 µL of streptavidine (1 µL in 10 mL blocking buffer). In Substrate control wells, 100 µl of blocking buffer was added. This was again followed by incubation for one hour at 37°C while shaking and washing for 5 times (PBS, 0.05%). While incubating, ortho phenylenediamine (OPD) was prepared in a dark recipient by dissolving two tablets in 12 mL of H2Od. To this solution, also 2.5 µg H2O2 was added. 100 µL of the prepared solution was added to all wells. Finally, the plates incubated for 15 min. at 30°C in the dark, whereafter the reaction was stopped by addition of 50 μL H2SO4 (4N) in each well. The plates were read at 492 nm. (max. absorption measure) and 655 nm. (background measure) by the Multiskan EX Microplate Photometer (Thermo Scientific). The intensity of the measured color (OD) reflects the amount of labeled antibody present in each well. After checking whether the two duplicate wells of positive and unknown stool samples roughly gave the same OD, the average OD was calculated for every sample. The cut off value for samples was fixed at 0.97 (determined during previous studies). Ratios per sample were calculated as follows: Ratio = average OD/cut off. When ratio was >1, samples were considered TS positive with a certainty of 99.9 %. A Microsoft Office Excel file was available to assist with these calculations. The ethanol Falcon tubes of positive samples were transported to Belgium upon confirmation by copro- PCR (in house PCR-RFLP).

3.2.6.7 Data Analysis

Quantitative biomedical data on the Pig Dissection forms, the pig sampling and the human sampling was double entered by two independent persons using the EpiCollect application on tablets. After checking data consistency, the data analysis was performed in R, using logistic regression analysis. The analyses looked into changes within each study arm before and after the intervention and between the E-arm and N-arm after the intervention. A significance level of 5% was used. The primary outcome was PCC as determined by necropsy and Ag ELISA on pig serum samples. The secondary outcome was human TS as determined by copro-Ag ELISA.

3.3 Results and Discussion

3.3.1 Coverage of the pig and human interventions

The coverage of both vaccination and OXF treatment of eligible pigs was sufficiently high, as can be seen in Table 12. The vaccination coverage decreased in the 4th intervention. The lower coverage and the noticeable discrepancy between vaccination and treatment coverage is due to lower consent rates for vaccination compared to OXF treatment. The more invasive injection method of vaccination, compared to the OXF drench, did not convene all pig farmers. Moreover, some of them believed that injection could cause the spread of ASF. Secondly, we noticed that the number of pig-keeping HHs and the total number of pigs has been reducing since the beginning of the study. High mortality rates and high turnover rates among pigs were probably due to ASF.

48

Table 12. Pig coverage data based on the six intervention databases (IE#1 - IE#6). * The number of vaccinated pigs may be lower than the number of oxfendazole (OXF) treated pigs as some owners refused vaccination (but allowed treatment).

Study time point Number of HHs that own pigs

Total number of pigs

Number of eligible pigs

Number of vaccinated pigs,

coverage

Number of treated pigs (OXF)*, coverage

Baseline (Oct’ 15) 58 184 NA NA NA

IE#1 (March ‘16) 40 153 103 99 (96.12%) 99 (96.12%)

IE#2 (July ‘16) 31 186 75 74 (98.67%) 74(98.67%)

IE#3 (Nov’ 16) 13 53 25 25 (100%) 25 (100%)

IE#4 (March ‘17) 11 62 37 24 (64.86%) 37(100%)*

IE#5 5 (July ‘17) 13 38 19 19 (100%) 19 (100%)

IE#6 (Nov’ 17) 12 72 47 45 (95.74%) 47* (100%)

For sufficient protection against the establishment of T. solium cysticerci, at least two vaccinations within four months (or within two consecutive interventions in this project) are necessary. In Table 13. we can remark that among vaccinated pigs, only a low number received their second shot.

This is due to the high pig mortality among the pig population due to ASF, the sales of pigs, the importation of new pigs and the vaccination of pigs that were not eligible in the previous intervention. Reluctance of pig farmers towards vaccination caused a lower coverage and the high pig turnover prevented the pigs from obtaining full protection. Both factors caused the vaccination program to be difficult to implement in the rural Zambian areas during this study and might cause similar difficulties in future implementation attempts. Table 13. Overview of the number of vaccinations at the six interventions (IE#1 - IE#6).

In Table 14. we see that the human treatment coverage (PZQ/NCZ) of eligible people in the E-

arm was relatively stable and sufficiently high during the two-year intervention period. The highest coverage is noticed at the first intervention (96%), the lowest at the second intervention (92%). An increase in total inhabitants is noticed, while the percentage of eligible human was stable. This is due to a high number of newborn, ineligible children and a number of children that turned five and became eligible for treatment. Reasons for non-treatment where refusal, illness or absence. Table 14. Human coverage data based on the six intervention databases (IE#1 - IE#6).

Study time point Total number of human

Number of eligible human, % of total

Number of treatments, % of eligible

IE#1 (March ‘16) 1078 897 (96) 857 (96)

IE#2 (July ‘16) 1091 916 (92) 846 (92)

IE#3 (Nov’ 16) 1134 966 (94 904 (94)

IE#4 (March ‘17) 1147 962 (93) 893 (93)

IE#5 5 (July ‘17) 1136 956 (94) 895 (94)

IE#6 (Nov’ 17) 1135 970 (95) 932 (95)

Study time

point

Number of

vaccinated pigs

Number of

pigs receiving their first

vaccination

Number of

pigs receiving their second vaccination

Number of

pigs receiving their third

vaccination

Number of

pigs receiving their fourth vaccination

Number of

pigs receiving their fifth

vaccination

IE#1 (March ‘16) 99 99 0 0 0 0

IE#2 (July ‘16) 74 52 22 0 0 0

IE#3 (Nov’ 16) 25 17 7 1 0 0

IE#4 (March ‘17) 24 11 12 1 0 0

IE#5 5 (July ‘17) 19 8 2 8 1 0

IE#6 (Nov’ 17) 45 39 1 2 2 1

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3.3.2 Dissection results

3.3.2.1 Overall dissection results

A total of 58 pigs were selected and purchased for dissections (E-arm, n = 26; N-arm, n = 32). All

pigs dissected were local breeds indigenous to the Eastern Province of Zambia. Cysticerci were detected by dissection in a total of 17 carcasses (29%). One out of 26 pigs (4%) in the E-arm and 16 out of 32 pigs (50%) in the N-arm tested positive. Of the 17 infected pigs, 11 were detected during the dissection of the first half of the carcass. An additional 6 cases were detected only during the dissection of the second half (full carcass dissection). Tongue palpation detected four out of the 17 (24%) dissection positives, of which three in the N-arm and one in the E-arm. Of these four, three were also positive during earlier blood sampling tongue palpation (Table 18.). T. hydatigena cysticerci were identified in four carcasses, representing 7% (4/58) of the total dissected carcasses. Three of the T. hydatigena postitives were found in the E-arm and one in the N-arm. Of the four T. hydatigena positives, one carcass was co-infected with T. hydatigena and T. solium. The other three carcasses (5%) had only T. hydatigena cysticerci. In 9 carcasses (16%), lungworms of the suspected Metastrongylus elongatus type was found. More details of the pigs found positive during post-intervention dissections can be found in Appendix 5.

3.3.2.2 Infection intensity

The number of cysticerci per carcass dissected ranged from one to 1061. A high proportion of infected carcasses (65%) had low to moderate infection levels (53% with ≤10 cysticerci and 12% with 11–50 cysticerci). High infection levels of ≥100 cysticerci were observed in 23% of infected carcasses (Table 15.). The majority of these small cysticerci had intact

walls, semitransparent membrane and transparent cysticercus fluids (Figure 9.). Compared to the

baseline dissection (Chembensofu et al., 2017), more carcasses had light (≤10 cysticerci) infections.

Table 15. T. solium infection levels in infected carcasses in the post-elimination (PE) and baseline (BL) survey.

Number of cysticerci in carcass Number of carcasses (%)

BL PE

≤10 16 (42) 9 (53)

11-50 13 (34) 2 (12)

51-99 1 (3) 2 (12)

≥100 7 (18) 4 (23)

≥10,000 1 (3) 0 (0)

Total 38 (100) 17 (100)

3.3.2.3 Distribution and classification of T. solium cysticerci

In 5 (29%) of the 17 infected carcasses only viable cysticerci were detected, in one (6%) only degenerated cysticerci were detected and in 6 (35%) only calcified cysticerci were detected. In 5 (29%) of the infected carcasses, cysticerci of various developmental stages were observed in the same carcass, and all three stages were present in two (12%) carcasses. The distribution and number of T. solium cysticerci in different locations are shown in Table 16. An estimated total of

Figure 9. Collection of T. solium cysticerci in a PBS Petri dish. Most of them have intact walls, semitransparent membrane and transparent cysticercus fluids. The scolex of the cyst in front can be noticed.

50

1764 cysticerci was detected in the different tissues and organs of the 17 T. solium infected

carcasses. Of the 1764 cysticerci detected, 1516 (86%) were viable, 74 (4%) were degenerated and 174 (10%) were calcified. In 7 carcasses, cysticerci were localized only in one organ or tissue (front leg, liver (two carcasses), neck, hind leg (two carcasses), tongue). The front leg recorded the highest number of T. solium cysticerci (408), followed by the hind leg (345), the neck (168)

and the tongue (161). The brain recorded the lowest number of cysticerci (6), however all of them were viable. A high percentage of infectious, viable cysts was also found in the neck (93%), the masseter (91%), the hind leg (91%) and the front leg (90%). However, transmission due to consumption of the brain or the masseter muscles is less likely, as these are only consumed in the Zambian culture when boiled, in contrast to the other parts of the carcass. The heart recorded the lowest percentage of viable cysticerci (0%) and the highest percentage of degenerated cysticerci (92%). The diaphragm, (707%), the tongue (77%) and the liver (79%) recorded also relatively low percentages of viable cysticerci. The tongue (Figure 10.) recorded the highest

percentage of calcified cysticerci (23%). 287 (16%) cysticerci were recorded in other than the predetermined places on the dissection sheets.

Table 16. Distribution and stage of the total number of T. solium cysticerci detected in the different muscles/organs in 17 infected carcasses.

Muscle/organ Cysticercus stage Total cysticerci/organ (%)

Viable Degenerated Calcified

Masseter 117 9 2 128 (7.26)

Heart 0 48 4 52 (2.95)

Tongue 124 0 37 161 (9.13)

Psoas 58 0 10 68 (3.85)

Diaphragm 16 2 5 23 (1.30)

Liver 93 5 20 118 (6.69)

Brain 6 0 0 6 (0.34)

Neck 157 3 8 168 (9.52)

Front leg 369 0 39 408 (23.13)

Hind leg 315 3 27 345 (19.56)

Other 261 4 22 287 (16.27)

Total (%) 1516 (85.94) 74 (4.20) 174 (9.86) 1764 (100%)

Most of the infected carcasses (10 out of 17) had cysticerci in the hind leg. Eight of the 17 infected carcasses had cysticerci in other places including the longissimus muscle (6; 35%), the intercostal muscle (5; 29%), the abdominal muscle (3; 18%), the lungs (3/17; 18%), the eyes (1; 6%), the spleen (1; 6%) and the muscles of the head (1; 6%) (Table 17.).

Figure 10. Dissected tongue. The frontal 3 mm slice contains three viable T. solium cysticerci.

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Table 17. Number and percentage of infected carcasses with T. solium cysticerci per organ.

Muscle/ organ Infected carcass/organ (%)

Masseter 4 (23.53%)

Heart 3 (17.65%)

Tongue 7 (41.18%)

Psoas 7 (41.18%)

Diaphragm 5 (29.41%)

Liver 8 (47.06%)

Brain 3 (17.65%)

Neck 8 (47.06%)

Front leg 8 (47.06%)

Hind leg 10 (58.82%)

Other 8 (47.06%)

Total (%) 17 (100%)

3.3.2.4 Ag ELISA

Of the 58 dissected pigs (E-arm, n = 26; N-arm, n = 32), a total of 6/58 (10%) tested positive for T. solium cysticerci circulating antigens by serum-Ag ELISA. Two out of 26 (8%) from the E-arm and four out of 32 (12.5%) from the N-arm. Of the four tongue positives, three tested positive on Ag ELISA (Table 18.). Of the 17 T. solium dissection positive carcasses, four serum samples tested

positive (24%)which is less than reported for the baseline dissections (68%) (Chembensofu et al., 2017). All of the detected had viable cysts. Of the 17 postives, 8 carcasses with viable cysticerci were detected. Of the carcasses containing viable cysts, four serum samples tested positive (50%). The sensitivity of the serum-Ag ELISA doubled if viable cysts were present, but was still bad (see 3.3.5, Table 20.). From the 41 T. solium dissection negative carcasses, Ag ELISA returned two positives, of which one was infected with T. hydatigena (based on dissection and PCR confirmation). Lack of specificity due to cross-reaction with T. hydatigena was described earlier by Devleesschauwer et al. (2013) and false positive results, infected with T. hydatigena were also present during the baseline dissections (Chembensofu et al., 2017). The other postive result might have been caused by a false positive Ag ELISA or by a false negative dissection result. In the article by Chembensofu et al. (2017) the occurrence of very small cysts (<3 mm) was described, which might have been present duringt the post-elimination dissections too. These small cysts are easily overlooked when applying the 3 mm slicing protocol, resulting in a false negative dissection result.

3.3.3 Blood sample results (Eligible pig population)

3.3.3.1 Ag ELISA

Of the total 106 sampled pigs (E-arm, n = 47; N-arm, n = 59), a total of 10/106 (9%) tested positive for T. solium cysticerci circulating antigens by serum-Ag ELISA. Three out of 47 pigs (6%) in the

E-arm and 7 out of 59 pigs (12%) in the N-arm tested positive. The percentage of positive blood sample in the E- and N-arm is comparable with the Ag ELISA results found for the smaller group of dissected pigs (3.3.2.4). More details of the pigs found positive during post-intervention follow-up sampling can be found in Appendix 6.

52

3.3.3.2 Tongue palpation

Of the 106 sampled pigs, a total of 6/106 pigs (6%) was found positive on tongue palpation, of which no pigs in the E-arm (0.00%) and 6 pigs in the N-arm (10%). By coincidence, all 6 tongue positive pigs were dissected later on. On dissection, only three out of the 6 pigs were positive by tongue palpation plus one extra. Four out of the 6 tongue palpation-positive pigs tested positive on Ag ELISA. Tongue palpation was not recorded for one pig in the E-arm (Table 18.). Table 18. Number of cyst and OD ratios for pigs found positive on tongue palpation during the post-intervention follow-up sampling and pig dissection. * positive on Ag ELISA

Pig ID number Pig dissection/sampling

Number of cysts Number of viable cysts

OD ratio

HER-P-004* Both 689 689 32.05

HER-P-005* Pig sampling 95 93 16.47

HER-P-009* both 670 670 34.02

HER-P-012 Pig sampling 66 0 0.93

HER-P-013 Pig sampling 23 0 0.95

HER-P-024* both 1061 963 31.65

NYE-P-021 Pig dissection 1 0 0.73

The PCC prevalence identified by tongue palpation during the post-elimination sampling survey for the E- and N-arm (respectively 0% and 10%) is comparable with the prevalence found by tongue palpation in the dissected pigs for the E- and N-arm (respectively 4% and 9%) (see 3.3.2.1). Both are a severe underestimation compared to the prevalence found by dissection of the carcasses (4% for the E-arm and 50% for the N-arm). Only 6 of the 17 dissection positives could be identified by tongue palpation during sampling, resulting in a sensitivity of 35% and four out of 17 during dissections, resulting in a sensitivity of 24%. A low sensitivity was also found at

baseline dissections (10%) (Chembensofu et al., 2017). Dorny et al., 2004) stated earlier that tongue palpation is a method of low sensitivity, which is only able to identify highly infected animals. All of these pigs that were tongue positive during the sampling had moderate to high infections (≥ 10 cysticerci) during the dissection later on (Table 18.). Moreover, these pigs had higher average OD

ratios compared to the pigs found positive trough serum-Ag ELISA, but negative on tongue palpation (Figure 11.). By consequence, tongue palpation is a useful method for primary surveillance in highly endemic regions areas, as areas where more than 10% of the pigs have cysts on their tongues showed a seroprevalence of at least 30% (Guyatt and Fevre, 2016). Although, the sensitivity of the method drops once the infection intensities decrease. No explanation was found for the different number of tongue palpation positives during sampling and dissection

3.3.4 Stool sample results

A total of 484 stool samples (E-arm) were collected and prepared for copro-Ag ELISA. Preliminary

results show a total of 11 (2.27%) human positive for antigen in the stool (TS positive).

19,34

7,43

Tongue + Tongue -

Average OD ratio

Figure 11. difference in average OD ratio of tongue positive and tongue negative pigs that were positive on serum-Ag ELISA.

53

3.3.5 Impact of the intervention on the PCC prevalence and TS prevalence Table 19. PCC/TS prevalence data. PME: * One pig had a calcified cyst in the tongue. ** in 2 of these pigs, cysts still need to be confirmed using PCR. *** Preliminary results. In the Negative Control study area, there were significantly more T. solium positive pigs at dissection than in the Elimination study area (OR = 25; p = 0.003). The TS prevalence dropped.

Study time point

Intervention arm

PORCINE CYSTICERCOSIS HUMAN

TAENIASIS

Blood samples PME Stool samples***

Blood

samples taken

Blood

sample positives

%

positive

PMEs

performed

Cyst

positive

%

positive % positive

Baseline (Oct’ 15)

Elimination 66 29 44% 37 17 46% 12%

Negative Control

102 42 41% 31 21 68% X

Post-

elimination survey (Jan’ ’18)

Elimination 47 3 6% 26 1* 4% 2%

Negative

Control 59 7 12% 32 16** 50% X

The PCC prevalence decreased from 46% to 4% in the E-arm and from 68% to 50% in the N-arm by means of pig dissections (Table 19.). By means of Logistic regression, there were significantly more T. solium positive pigs at dissection in the N-arm compared to the E-arm (OR=25; p=0.003),

meaning that the interventions did have a significant effect. The only cyst found in the E-arm was a calcified cyst in the tongue of one pig. As calcified cysts are not infectious, we can say that elimination of infectious T. solium in the pig population was reached. Nonetheless, no elimination

of the parasite transmission was reached as TS was not eliminated. We know that, due to ASF, the pig population decreased over the interventions and there was a high pig turnover, resulting in low vaccination protection and highly susceptible animals. The low final PCC prevalence suggests that susceptible untreated, newborn or imported pigs and treated pigs that did not receive two vaccination shots, did not (re)infect themselves, indicating that environmental egg contamination diminished. This could be due to a combination of less tapeworm carriers and the impact of the HE sessions, increasing the use of latrines. The serological results support the reduction in PCC prevalence as also a reduced number of positive blood samples was found. Positive samples decreased from 44% to 6% for the E-arm and also an unexpected big decrease from 41% to 12% was found for the N-arm. Especially for the N-arm a lower percentage of positive pigs was found during the post-intervention sampling survey (12%) compared to the percentage of positives found on pig dissections (50%). A possible explanation for the unexpected decrease on serum-Ag ELISA is a low sensitivity of the serum-Ag ELISA test, which is normally between 65% and 93% (Dorny et al., 2004). Considering the full carcass dissections as gold standard, only 4 of the 17 infected pigs by pig dissections were detected by the Ag ELISA of the dissected pigs, rendering a sensitivity of 24% (see 3.3.2.4). A sensitivity of 24% is low compared to values mentioned in the literature and compared to the sensitivity of the test (68%) found at baseline dissections (Chembensofu et al, 2017). In turn, a low sensitivity of the Ag ELISA might be caused by the presence of a low amount of circulating antigen in the blood. The circulating antigen in a carcass that contains a low number and moreover calcified or degenerated cysts is lower compared to a carcass that contains a high number of viable cysts. Consequently, serum samples of these carcasses are often not recognized by the Ag ELISA that is coated with monoclonal antibodies made upon the recognition of antigen of viable cysts, resulting in false negative results (Brandt et al., 1992). At the baseline studies, this point

54

was proven as the sensitivity of the serum-Ag ELISA improved when an infected carcass contained more and more viable cysts (Chembensofu et al., 2017) and this is also true for the post-elimination study, however, the overall test performance was lower in all of the groups (Table 20.) The percentage viable, degenerated and calcified cysts was respectively 86%, 4% and 10% of the total number (1764) of detected T. solium cysticerci (Table 16.), which is identical to the

percentages found at the baseline intervention (Chembensofu et al., 2017). However, when looking at the 17 individually infected pig, only 53% had a cyst load of 10 or more cysts compared to 58% at baseline, only 47% had at least one viable cyst compared to 58% at baseline and only 29% had 10 or more viable cysts compared to 34% at baseline (Table 20.). This implies that the

percentage of pigs with no or less than 10 viable cysticerci, was higher at the post-elimination survey than at the baseline survey (respectively 71% and 66%) and more carcasses had a low (<10 cysticerci) infection load (47% at baseline compared to 42% at baseline). Nevertheless, these relatively small differences do not explain the bad performance of the test. Table 20. The number (n) and percentage of carcasses with 1/10 (viable) cysts and the test performances of the Ag ELISA according to the infection level and cyst stage in 17 or 38 confirmed infected pigs compared in the baseline (BL) and post-elimination (PE) survey.

In Figure 12. a boxplot for viable,

degenerated and calcified cysts is shown for the pigs found positive on both dissection and serum-Ag ELISA and the group of pigs found positive on dissection but negative on serum-Ag ELISA. A significant effect (p=1.11x105) was found between the number of viable cysts and the outcome of the serum-Ag ELISA. No significant effect was found between the amount of degenerated or calcified cysts and the outcome of the serum-Ag ELISA, however, the sample size was too small (n1=4, n2=4). Figure 12. and Table 19. confirm the

influence of the number of (viable) cysticerci on the sensitivity of the Ag ELISA,

The part of PPC prevalence reduction found in the blood sampling results of the N-arm that is not explained by a low sensitivity of the Ag ELISA and which is also present in the necropsy results might be due to the high

T. solium infection level and stage Intervention n/% Ag ELISA+ Ag ELISA- Sensitivity (%)

carcasses with ≥ 1 cysticercus PE 17/100 4 13 24

BL 38/100 26 12 68

carcasses with ≥ 10 cysticerci PE 9/ 53 4 5 44

BL 22/58 20 2 91

carcasses with ≥ 1 viable cysticercus PE 8/ 47 4 4 50

BL 22/58 20 2 91

carcasses with ≥ 10 viable cysticercus PE 5/ 29 4 1 65

BL 13/34 13 0 100

Figure 12. Boxplot of the amount of viable, degenerated and calcified cysts for serum-Ag ELISA positive (+) and negative (-) groups of the positive dissected pigs. Also the average is shown (Av.).

55

morbidity caused by ASF. Mortality of the host might have caused the parasite prevalence to drop. Moreover, minimal HE interventions were also given in the N-arm, which might have had an effect too. TS prevalence decreased from 12.40% to 2.27% (Table 19.), meaning that the interventions

succeeded in decreasing the number of tapeworm carriers, however not in eliminating them. As the human treatment coverage did not reach 100%, the possibility exists that some tapeworm carriers were not treated during the entire two-year intervention period. The life span of the adult tapeworm is estimated at one to five years (Garcia et al., 2014), meaning that tapeworm carriers at the baseline survey that did not take treatment might have been re-detected. Moreover, some people could have re-infected themselves after treatment. We did not trace back the infection source of the infected people, so reinfection might have occurred in a neighboring village, where PCC prevalence is suspected to be similar to the E-arm baseline prevalence (>40%).

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4. Conclusion

Next to a social and economic burden, the complex disease caused by T. solium is related to a

total of 2.8 million DALYs each year (WHO, 2015a). This number might even be an underestimation as it is based on epilepsy and no headaches or other symptoms caused by cysticercosis were included in the calculation. Despite justified global attention, there is still no validated strategy available to tackle the number one foodborne parasitic disease (WHO/FAO, 2014; WHO, 2017), even though this was a 2015 target included in the 2012 road map to tackle the NTDs (WHO, 2012).

Nevertheless, new evidence on the control and elimination was identified in the first part of this thesis, the systematic review. Since 2014, MMs are used more than ever to render quick insights on the effect of several interventions. Three MMs included in the review, agreed that single interventions have the ability to control the parasite (Braae et al., 2016a; Johansen et al., 2017; Winskill et al., 2017). Among them, highest reductions in TS/PCC/HCC prevalence were obtained through chemotherapeutic interventions to humans or pigs. However, coverage was often mentioned as a limiting factor for the effectiveness of this strategy, especially for pigs. A frequently suggested solution to this limitation is a combined human and pig treatment, a combination of pig treatment and vaccination or a combination of any therapeutic treatment with a long-term strategy. Combinations or multiple rounds were also pointed out as necessary to obtain sustainable effects. On the human side, track and treat of TS cases was modelled as a very effective strategy in the MM by Winskill et al. (2017), but is currently hypothetical due to the lack of sensitive, field-ready tests and the possible adverse effects of PZQ. If ongoing research on development of better diagnostic tools is successful, future field studies may confirm the effectivity of human track and treat and provide an additional cost-efficient strategy. Human MDA is feasible at the moment and effective in reducing TS/PCC/HCC prevalence, but lacks a sustainable effect if applied once. Furthermore, most people are unnecessarily treated, which implies the waste of resources and unnecessarily exposing people to adverse effects. To increase sustainability, multiple treatment rounds were suggested by MMs and supported by a field study (Braae et al., 2017). An increase in cost-efficiency might be obtained by incorporating TS treatment into larger NTDs or STH programs, as measured by two field studies (Ash et al., 2015; Braae et al., 2016b), but the lack of detailed co-distribution-maps might hamper effective co-treatment. Ring-strategies, treating residents within 100 m. of a heavily infected pig also increase the cost-efficiency compared to human MDA and seem promising (O'Neal et al., 2014). Nevertheless, the results are preliminary and controlled, randomized studies should be conducted in large populations to confirm efficacy. At last, some field studies only targeted part of the population (SAC) (Braae et al., 2016b; 2017), but the efficacy of this method lacks evidence. On the pig side, strategies including pig treatment with OXF are often hampered by insufficient coverage. Lightlowlers et al. (2017) therefore suggested a three-monthly combined treatment and vaccination (TSOL18) scheme for pigs, which would realize protection of all slaughter-aged pigs within ten months. As such, transmission to humans, through consumption of these pigs, would be prevented. An experimental pig study discovered that also intervals up to 20 weeks between two vaccinations provide adequate antibody protection (Lightowlers et al., 2016a). However, this strategy has not been tested in the field and sufficient coverage is doubtful as there is a fast turnover in pigs. Moreover, there are some practical constraints to the implementation of a vaccination scheme such as the storage and transport of the vaccine under appropriate cold chain conditions, the practical implementation of vaccination as well as the compliance of the pig owners. Integration of T. solium vaccination in another disease control program, that is perceived

57

as more economically relevant by farmers (for example: ASF), might increase interest and compliance towards any intervention. However, this has not been described yet. The quick impact of the combination of human and pig treatment as predicted by the MMs was confirmed in a South Asian context by the study of Okello et al. (2017) and Garcia et al. (2016). Moreover, elimination of transmission through a one-year intensive program, combining human and pig treatment/vaccination. was reached by Garcia et al. (2016), even though this was measured to be unlikely by most of the MMs. In the N-arm of the CYSTISTOP study, there were significantly more T. solium positive pigs at dissection compared to the E-arm (OR=25; p=0.003). In addition, an important reduction in occurrence of T. solium was achieved in the E-arm after the two-year integrated intervention

approach. The applied strategy was comparable with the strategy used in Garcia et al. (2016). Positivity at pig necropsy in the E-arm lowered from 46% at baseline to 4% post-intervention and the one pig that was found positive at necropsy had one calcified cyst. As calcified cysts are not infectious, the elimination of infectious T. solium in the pig population was reached. Full elimination of transmission did not succeed, as human TS cases had greatly reduced, but not to zero. The main problems encountered were the strong decline and the high turnover in the pig population (presumed due to ASF), whereby very few pigs received the required two vaccinations, and even less received two vaccinations within four months, once more confirming that a vaccination program in Zambian rural areas, is difficult to implement. The treatment of pigs was more acceptable to the farmers (drench versus injection, with the latter being linked to transmission of ASF) and would therefore be more sustainable. The study sites will be followed for another three years, whereby the sustainability of the short-term intensive strategy will be measured through active monitoring. This active monitoring will include six-monthly human stool samplings, porcine blood samplings and treatment of all people (PZQ) and pigs (OFZ) within a 50 m. radius around the detected human TS or PCC case. Both studies (Garcia et al., 2016; CYSTISTOP, 2018) show that elimination of transmission in the pig population is an achievable target. Elimination of TS or transmission of the parasite was not reached in the CYSTISTOP study and not measured by Garcia et al. (2018). Nevertheless, MMs do not give an unambiguous answer whether elimination is reachable, assuming that it will not be easy and area-dependent factors might play a big role. Moreover, the intensity of the strategy raises the question whether elimination is a practical and economically viable target and which other measures are needed to sustain the effect.

Short-term elimination goals might not be suitable for the low and middle income endemic countries like Zambia, as they are too expensive and there is no framework that can supply and apply these measures. A stepwise approach, consisting of initial or regular therapeutic treatment, potentially implemented as a ring-strategy or integrated in another NTD/STH program, with additional long-term measures seems a more reachable strategy. Transmission is often associated with an environment and habits that support transmission of the parasite. For example, poverty, lack of sanitation, free-range pig husbandry, a lack of sufficiently sensitive meat inspection and a lack of encouragement around safe consumption are factors that might take more than one generation to alter. With this in mind, investing in long-term strategies might provide broad-based knowledge, habits and hygiene that have advantages far beyond that of T. solium

and provide structures and knowledge that are passed on to next generations. Long-term strategies might also be the key in keeping transmission low, after control or elimination of the parasite is reached. The provisions of adequate sanitation and programs addressing sanitation related taboos are one of these measures (Bulaya et al., 2015; Thys et al., 2015). HE on the parasite itself, hygienic measures, pig confinement, symptoms and handling/cooking of pork meat

58

is another one. Recently, more scientifically based educational programs and tools became validated and available. ‘The Vicious Worm’ for example, is an easy to understand, computer-based program that was successful in improving knowledge in both professionals (Ertel et al., 2017) and children (Hobbs et al., 2018). Therefore, it provides a cheap and easy to adapt method to reach different stakeholders across sectors, including children. The PRECEDE-PROCEED model (Ngowi et al., 2017; Carabin et al.,2018) is another science-based HE model that rendered area specific community-engaged participatory interventions. The study obtained a reduction in cumulative incidence and prevalence of active HCC, making it one of the first HE studies that measured an effect on prevalence/ incidence of T. solium. Additional to a stepwise approach, collaboration with veterinary and medical services for active surveillance and meat inspection in endemic areas is necessary and might prevent migration of infective pigs and humans and sustain the effect of any intervention.

Lastly, implementation of any intervention measure depends on the willingness of policy makers. Therefore, it is important to provide actual data on the prevalence and burden of T. solium and

communicate this data at governmental level. Moreover, it is necessary to provide data on effective measures on the control of T. solium that are within reach, meaning financially and

practically sustainable for a particular country or region.

59

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Appendix 1.

Pig dissection sheet

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Appendix 2.

SOP_Pig dissection

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Appendix 3.

SOP_Pig euthanasia

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Appendix 4.

SOP_Blood Sampling Pigs

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Appendix 5.

Details of pigs that were T. solium positive during the post-intervention dissections.

Pig code

Household

code Study arm

Number of

vaccinations

Total number of T. solium

cysticerci

PCR result T.

solium ELISA ratio

HER-P-002 ROB014 Negative 0 1 positive 0.73

HER-P-004 CHU054 Negative 0 689 positive 32.05


HER-P-007 KAP051 Negative 0 10 positive 0.65




HER-P-014 CHY011 Negative 0 4 positive 0.69



HER-P-018 HER027 Negative 0 3 positive 0.61




HER-P-030 ETA006 Negative 0 1 to be confirmed 0.80

HER-P-032 KAP051 Negative 0 124 to be confirmed 0.77

NYE-P-021 MTA032 Elimination 0 1 positive 0.73

Appendix 6.

Details of ELISA-postive pigs during the post-intervention follow-up sampling.

Barcode Study arm HH code OD Ratio ELISA result

**HER-P-004* Negative CHU054 2.981 32.05 positive

**HER-P-005* Negative CHU054 1.532 16.47 positive

**HER-P-009* Negative KAP027 3.164 34.02 positive

**HER-P-024* Negative KAP027 2.595 31.65 positive

HER-P-053 Negative CHU067 0.292 3.56 positive

HER-P-058 Negative CHT005 2.034 24.80 positive

HER-P-061 Negative CHU069 0.468 5.71 positive

NYE-P-004* Elimination KAY014 0.099 1.06 positive

NYE-P-026* Elimination SAF005 0.084 1.02 positive

NYE-P-046 Elimination PET001 1.455 15.81 positive

* Pigs that were also dissected

** Pigs that were positive on dissection

control of taenia solium in zambia · taenia solium en behoort tot de ‘verwaarloosde tropische...

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