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Insights from experimentally infecting humans with hookworm

Insights from experimentally infecting humans with hookworm. Emeritus Professor Rick Speare School of Public Health, Tropical Medicine and Rehabilitation Sciences, James Cook University & Director, Tropical Health Solutions Townsville, Queensland, Australia 17 October 2012.

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Insights from experimentally infecting humans with hookworm

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  1. Insights from experimentally infecting humans with hookworm Emeritus Professor Rick Speare School of Public Health, Tropical Medicine and Rehabilitation Sciences, James Cook University & Director, Tropical Health Solutions Townsville, Queensland, Australia 17 October 2012 Presentation to Medical Research Council (Gambia) • Available at: http//www.tropicalhealthsolutions.com/hookworm

  2. Orientation - map Townsville Anton Breinl Centre for Public Health and Tropical Medicine

  3. Dr John Croese & Castle Hill

  4. Hygiene Hypothesis: STH style • Although soil transmitted helminths (STH) cause significant morbidity in developing countries, the lack of STH in developed countries is hypothesised to increase the incidence of allergic and autoimmune disease. • STH will induce a Th2 response against the parasite and this will be down-regulated by regulatory T cells which will also non-specifically down-regulate the destructive immune response (Th1) of the host that is responsible for allergic and autoimmune diseases.

  5. Relationship between prevalence of multiple sclerosis and STH Fleming & Cook. Neurology 2006;67:2085 • Trichuris trichiura as a marker of STH & low levels of community sanitation • Prevalence of MS & Tt in 35 countries • Correlational study

  6. Multiple Sclerosis vs Whipworm Fleming & Cook. Neurology 2006;67:2085 • Prevalence of MS falls steeply once threshold of 10% Tt is reached • This applied for marginalised populations sharing same country (Jews – Arabs in Israel) 10% Is the protective effect due to Tt or gut exposure to pathogens?

  7. Some worms are protective; some aren’t: Care is needed! • Meta-analysis of asthma and association with STH • STH in general no significant effect • Ascaris is associated with asthma • Hookworm is protective Leonardi-Bee et al Am J Resp Crit Care Med 2006;174:514

  8. Effect of anthelmintics on allergic reactions Skin reactivity to dust mite antigen increased after STH controlled • Van den Biggelaar et al J Inf Dis 2004;189:892

  9. Flohr et al. J Aller Clin Immunol 2006;118:1305

  10. How do parasites exert an effect? • Brown & Jackson Para Immunol 2004;26:429 Immune response

  11. Trichuris suis for IBD • Whipworm eggs collected from worms in experimentally infected pigs • Incubated, made aseptic • 2500 eggs orally every 3 weeks

  12. All open trials • Significant percent of remissions for both Crohn’s Disease and Ulcerative Colitis • No significant side effects 80% of patients benefited Summers et al. Gut 2005;54:87 Summers et al. Am J Gastrolenterol 2003;98:2034

  13. Necator americanus

  14. Life cycle • Skin penetration • Blood-lung migration • Occurs within 48 hrs PI • Juvenile worms arrive in smint in 4 weeks PI • Adults mature at 7-8 weeks PI

  15. N. americanus in gut • Attaches to mucosa of small intestine • Sucks blood • Little known about behaviour of NA in human gut since can not be directly observed

  16. Infective larvae (L3i)

  17. Harada-Mori Technique • 25°C • In dark • 7 days

  18. 12 hr post-infection

  19. 1 day post-infection

  20. 2 days post-infection

  21. Infection worked! • We inoculated 9 immunosuppressed Crohn’s disease patients and 3 normal donors with 15 to 101 L3i • All 9 Crohn’s patients and 2/3 donors developed a patent infection Insight: Imunosuppressed and normal hosts can be infected with Necator with no or minimal negative effects.

  22. What did the hosts think? • Human hosts loved their worms! Insight: Finding subjects to be inoculated with hookworms is not difficult.

  23. Some problems! • Penetration site • Small intestine

  24. 2 days post-infection

  25. 4 days post-infection

  26. 6 days post-infection

  27. 7 days post-infection

  28. 8 days post-infection

  29. 9 days post-infection

  30. 10 days post-infection

  31. 11 days post-infection

  32. 12 days post-infection

  33. 15 days post-infection

  34. 20 days post-infection

  35. Severe local response on reinfection Insight: Penetration of L3i is uneventful in most, worrying in some

  36. Why the severe local reaction?

  37. L3i are “dirty” • L3i are heavily contaminated with faecal bacteria. • Standard techniques of multiple washing do not reduce the contamination. • Is the hookworm sheath left on skin or does it remain in epidermis? • Washing in providone iodine for 15 min was added after harvesting. • Could L3i be made sterile?

  38. Better Quality L3i • Melissa Logan – BMedLabSc honours thesis Bacteria are associated with L3i

  39. In vitro tests

  40. In vivo tests: penetration • 15 L3i; 3 groups – 5 subjects per group

  41. Patency of infection • 100% patency for both techniques Antibiotic treated L3i were equivalent to standard L3i

  42. Conclusion • Treatment of L3i with bleach and antibiotics gives viable larvae of comparable potency. • These L3i are sterile. • Bleach and antibiotic technique should be the gold standard for L3i for experimental infection.

  43. Recommended Protocol • Harvest L3i and centrifuge • Place in 0.25% sodium hypochlorite for 10 mins • Wash twice using centrifugation in sterile distilled water to remove any residual sodium hypochlorite • Place in a solution of benzylepenicillin (180 mg/L) and ceftazadime (1.0 mg/L) for 60 mins • Centrifuge to remove excess solution • Wash L3i twice using centrifugation in sterile distilled water to remove any residual antibiotic solution. Are skin reactions less?

  44. Safety when working with L3i • For any item in the laboratory a Material Safety Data Sheet (MSDS) is needed. • MSDS important when L3i of N. americanus are being used in laboratory. • The MSDS for hookworm said kill spills of L3i with bleach (Health Canada 2001). • Did this actually work?

  45. What kills L3i rapidly? • Bleach did not kill L3i • Ethanol 70-100% did • Dettol killed L3i Insight: Current MSDS was inaccurate! A new one based on evidence was needed.

  46. New Hookworm MSDS • Speare et al. Australian Journal of Medical Science 2008;29:91-96. • Online at http://www.tropicalhealthsolutions.com/MSDSNecatoramericanus

  47. Abdominal pain • Hosts developed central abdominal pain about 3 weeks post-inoculation

  48. Healthy Volunteers: • 1 of 3: developed vigorous local reaction • 3 of 3: Central, non-colicky abdominal pain started about 3.5 weeks and persisted for about 5 weeks

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