Supplementary Appendix

Transcription

1 Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Garcia HH, Gonzalez AE, Tsang VCW, et al. Elimination of Taenia solium transmission in northern Peru. N Engl J Med 2016;374: DOI: /NEJMoa

2 SUPPLEMENTARY APPENDIX Table of Contents Page Author s contributions.. 2 Details of intervention procedures 3 Additional information on Phase I, II, and III control interventions.. 5 Table S1. Seroincidence of antibodies against cysticercosis in pigs by intervention strategy, Phase I. 6 Table S2. Phase II: Results of pig necropsy by serologic status and intervention strategy for pigs sampled immediately post-intervention 7 References. 8 1

3 AUTHORS CONTRIBUTIONS HHG, AEG, VCWT, FLZ and RHG designed the study, and a field team including GG, SR, LMM, VA, and AD gathered the data. AH and SO analyzed the data. The first author wrote the first draft, and all the coauthors made the decision to submit the manuscript for publication. No one who is not an author contributed to the writing of the manuscript. 2

4 DETAILS OF INTERVENTION PROCEDURES Antibody serology. Pig sera were assessed by enzyme-linked immunoelectrotransfer blot (EITB, Western blot) using seven purified glycoprotein T. solium antigens. 1 Antibody responses to at least one antigen are considered positive and demonstrate exposure or infection, with the likelihood of viable cyst infection increasing when antibody responses to multiple antigens are present. 2 Blood samples of 3 ml were collected by venipuncture from the cava vein of pigs. Stool examination. Whole stool samples were collected and examined macroscopically for the presence of tapeworm proglottids and/or scolices. Stool aliquots were then placed in 5% formol-phosphate Buffered Saline for subsequent analysis by light microscopy using standard ether sedimentation technique for detection parasite eggs and coproantigen ELISA. Examination of stools by light microscopy is poorly sensitive for detection of Taenia sp. eggs. Conversely, detection of tapeworm antigens by ELISA in stools is highly sensitive and allows the diagnosis of tapeworms even before they reach the patent, egg-producing stage. 3,4 This assay uses a rabbit polyclonal antibody against immature tapeworm antigens in a capture ELISA format. We modified the classic coproantigen assay by using capture antibody and conjugate specific to T. solium. 5 In Phases I and II, all stool samples were analyzed by both light microscopy and coproantigen ELISA. In Phase III, all stool samples were analyzed by the most sensitive method (ELISA coproantigen), but only a random selection of 30% of the samples were also examined by light microscopy. Niclosamide (NSM) treatment of humans. NSM is the drug of choice for T. solium taeniasis because it is not absorbed systemically and has minimal side effects. It can be safely used in regions endemic for NCC since it has no activity against larval stage cysts. NSM was administered in a single oral dose of 2 g in adults, 1 g in children older than 6 years, and approximately 50 mg/kg in children 2-6 years old. 6 The pills were carefully ground and mixed with fruit juice for ingestion. 3

5 Oxfendazole (OFZ) treatment of pigs. OFZ solution 22.5% was administered orally to pigs 1 month old in a single drench dose of 30 mg/kg. This regimen destroys all cysticerci in pig muscle although cyst degeneration takes >1 month. 7-9 Since no data on longevity of tissue residues was available, villagers were advised not to sacrifice or consume the pigs in the week following treatment. The withdrawal period for OXF has now been established, and future interventions should recommend against meat consumption until at least 17 days post treatment. 10 Necropsy of pigs. Detailed necropsy using fine dissection was performed since most infected pigs harbor very few cysts. 2 Pigs were purchased from villagers at slightly higher than market price, anesthetized and humanely sacrificed. The entire carcass including the brain was thinly sliced (3-4 mm cuts) and examined in detail looking for live, non-degenerated cysts (cystic structures filled with clear fluid) or dead cysts (cystic structures showing macroscopic changes suggestive of advanced degeneration, either collapsed or with colloid or semi-solid contents). Cyst evagination. We evaluated transmission potential of live, non-degenerated cysts by incubating them in a bile solution. 11 Cysts in which the scolex of the tapeworm emerged from the cyst wall and moved were considered capable of causing taeniasis if ingested. Cysticercosis (TSOL18) pig vaccine. The TSOL18 vaccine, developed at University of Melbourne in Australia, was produced and supplied for use in this program. 12,13 TSOL18 was intramuscularly administered in two doses spaced three weeks apart and in conjunction with other interventions. Quil A was used as adjuvant. Pigs 2 months old were immunized. Hog cholera vaccine. Hog cholera is endemic in most developing countries and kills approximately 20% of all the porcine population every year. We vaccinated pigs against Classic Swine Fever using a liveattenuated vaccine at no cost to improve villager compliance with the elimination program. Vaccination was applied at the same time with blood sample collection and OFZ treatment of the pigs. 4

6 ADDITIONAL INFORMATION ON PHASE I, II AND III CONTROL INTERVENTIONS Phase I Interventions: Phase I compared the effectiveness and acceptability of 6 intervention strategies in 42 villages. The first two strategies (Mass Treatment and Minimal Mass Treatment) applied mass chemotherapy with niclosamide in humans for taeniasis and oxfendazole in pigs for porcine cysticercosis using two different timing intervals; the third (Mass Screening), screening and treatment of taeniasis in humans and cysticercosis in pigs; fourth (Strategic Treatment), continuous treatment of pigs born into or entering the village; fifth (Prevention Education), disease prevention education for taeniasis/cysticercosis; and sixth (Pig Replacement), removal of the pig population and replacement with non-infected animals. Stratified randomization based on village size and baseline pig seroprevalence was used to assign strategies 1-5 to 36 villages, while 6 isolated villages were assigned by convenience to Pig Replacement. Phase II Interventions: The two most effective strategies from Phase I were selected and modified increasing the number of treatment rounds and by shortening the interval between treatments, and applied in 17 larger villages along the Western margin of the Tumbes River. There were four intervention groups (Mass Treatment or Mass Screening, either with or without pig vaccine). Village assignment was based on geographic area so that neighboring villages shared the same strategy. The Mass Screening strategy was modified by replacing pig screening with mass pig chemotherapy for logistical reasons, although screening for taeniasis in humans was still performed. Phase II Intervention: The final scaled-up intervention, mass treatment with vaccine, was applied in 107 villages covering a population of 81,170 people and a total of 55,638 pigs. Niclosamide was administered to 48,099 people, 54,262 people and 55,767 people respectively in rounds 1-3, with 84.7% of the entire population receiving at least one dose. For pigs, oxfendazole was given every 2 months and two vaccination campaigns of two rounds each were also performed. A total of 81,977 doses of oxfendazole and 49,224 doses of TSOL18 vaccine were administered. 5

7 Table S1. Phase I: Seroincidence of antibodies against cysticercosis in pigs by intervention strategy Minimal Mass Mass Strategic Mass Prevention Treatment Treatment Treatment Screening Education Mean pig age at 9.47 (7.70) 9.22 (7.44) 8.38 (5.75) 8.76 (7.09) 9.02 (6.75) baseline, months (sd) Incidence*, (95% CI) 5.00 ( ) 4.11 ( ) 4.77 ( ) 4.02 ( ) 5.17 ( ) Crude incidence ref ratio**, (95% CI) 0.80, , , , 0.94 Age-adjusted incidence ratio**, (95% CI) , , , , 0.95 ref * An incident event was defined as a pig having 3 or more reactive bands on EITB LLGP after having 0,1 or 2 reactive bands in a prior round. Incidence is reported as the number of new events per 100 pigmonths. ** Poisson regression with Prevention Education as the reference group. 6

8 Table S2. Phase II: Results of pig necropsy by serologic status and intervention strategy for pigs sampled immediately post-intervention. EITB LLGP Pig blood sample Pig necropsy sample Pigs with live, nondegenerated cysts** no. of bands no. no. % no. %. Mass Treatment with vaccine Seronegative bands bands bands Total Mass Treatment without vaccine Seronegative bands bands bands Total Mass Screening with vaccine Seronegative bands bands bands Total Mass Screening without vaccine Seronegative bands bands bands Total

9 REFERENCES 1. Tsang VC, Pilcher JA, Zhou W, et al. Efficacy of the immunoblot assay for cysticercosis in pigs and modulated expression of distinct IgM/IgG activities to Taenia solium antigens in experimental infections. Vet Immunol Immunopathol 1991;29: Gonzalez AE, Lopez-Urbina T, Tsang B, et al. Transmission dynamics of Taenia solium and potential for pig-to-pig transmission. Parasitol Int 2006;55 Suppl:S Allan JC, Avila G, Garcia Noval J, Flisser A, Craig PS. Immunodiagnosis of taeniasis by coproantigen detection. Parasitology 1990;101 Pt 3: Guezala MC, Rodriguez S, Zamora H, et al. Development of a species-specific coproantigen ELISA for human Taenia solium taeniasis. Am J Trop Med Hyg 2009;81: Bustos JA, Rodriguez S, Jimenez JA, et al. Detection of Taenia solium taeniasis coproantigen is an early indicator of treatment failure for taeniasis. Clin Vaccine Immunol 2012;19: Letter TM. Drugs for Parasitic Infections. New Rochelle, NY Gonzales AE, Garcia HH, Gilman RH, et al. Effective, single-dose treatment or porcine cysticercosis with oxfendazole. Am J Trop Med Hyg 1996;54: Gonzalez AE, Falcon N, Gavidia C, et al. Treatment of porcine cysticercosis with oxfendazole: a dose-response trial. Vet Rec 1997;141: Gonzalez AE, Falcon N, Gavidia C, et al. Time-response curve of oxfendazole in the treatment of swine cysticercosis. Am J Trop Med Hyg 1998;59: Moreno L, Lopez-Urbina MT, Farias C, et al. A high oxfendazole dose to control porcine cysticercosis: pharmacokinetics and tissue residue profiles. Food Chem Toxicol 2012;50:

10 11. Cañedo L. Evagination of the metacestode of Taenia solium. In: Flisser A, Willms, K, Laclette, JP, Larralde, C, Ridaura, C, Beltran, F, ed. Cysticercosis: Present State of Knowledge and Perspectives. New York: Academic Press; 1982: Flisser A, Gauci CG, Zoli, A, et al. Induction of protection against porcine cysticercosis by vaccination with recombinant oncosphere antigens. Infect Immun 2004;72(9): Gonzalez AE, Gauci CG, Barber D, et al. Vaccination of pigs to control human neurocysticercosis. Am J Trop Med Hyg 2005;72: