1. Clinical and Epidemiological Aspects of Escherichia coli O157:H7 in Latin America Alejandro Cravioto, M.D., Ph.D Rosario Morales, M.D., Ph.D Armando Navarro, B.Sc., M.Sc. Faculty of Medicine, UNAM Mexico City

2. Dr. Alejandro Cravioto (b. Mexico City, 1947) In 1973, Dr. Cravioto received his Medical Degree with honors from the Faculty of Medicine at the National Autonomous University of Mexico. Between 1973 and 1976, he continued his studies in Pediatrics at the National Institute of Pediatrics in Mexico City. In 1977, he received a Diploma in Tropical Public Health, and in 1981, a Ph.D. from the London School of Hygiene and Tropical medicine at the University of London.

3. Escherichia coli The organism – Gram negative, non-spore forming rod, Fam. Enterobacteriacea (aerobic/facultatively anaerobic) Serotypes – O, H & K antigens Toxins - ETEC produce LT and/or ST - EHEC produce Stx toxins

4. Generally harmless Pathogenic groups - Enteropathogenic (EPEC) –Enterotoxigenic (ETEC) –Enterohemorrhagic (EHEC) – HC, HUS, TTP –Enteroinvasive (EIEC) –Enteroaggregative (EAEC) –Diffusely adherent (DAEC)

5. E. coli O157:H7 1982 – First recognized as a pathogen 1985 – Associated with hemolytic uremic syndrome 1990 – Outbreak from drinking water 1991 – Outbreak from apple cider 1993 – Multi-state outbreak from fast food hamburgers 1995 – Outbreak from fresh produce 1996 – Outbreak in Japan – Multi-state outbreak from unpasteurized apple juice

6. Outbreaks associated with E. coli O157:H7 in food products Food productCountry

7. E. coli O157:H7 - incubation – 3 to 9 days - bloody diarrhea, renal failure, thrombocytopenia - infective dose – small to large number of organisms - the young & the elderly - dialysis, fluid balance, hypertension

8. Pathogenesis - EHEC (O157:H7) – HC, HUS & TTP - Causes adherence & effacing lesions on intestinal cells (HC) - Systemic complications (HUS) – acute renal failure - toxin causes death of endothelial cells of glomeruli & afferent arterioles, narrowing of blood vessels, hemolytic anemia & thrombocytopenia, reduced glomerular filtration & kidney tissue necrosis

11. Incidence of E. coli O157 infection reported by laboratories (per 100 000 population)

12. Outbreaks of enterohemorrhagic E. coli reported in Japan

13. Incidence of hemolytic-uremic syndrome in children 4 years of age or younger (per 100 000 population)

14. Comparison of EHEC isolation rates from HUS patients in North America, Europe, and South America a Number of children with EHEC / number of children with HUS whose stool cultures yielded E. coli (%).

15. Argentina has one of the highest recorded HUS rates (300 cases/year) in Latin America The risk of HUS in Stx-EC associated bloody diarrhea is about 4-5%, with 14% of children developing incomplete HUS A total of 80% of Stx-EC isolated from patients with diarrhea belonged to non- 0157 serogroups such as O26, O91, 0103, O111, O113, O128, O145

16. Incidence of Shiga-toxin (Stx)-associated illness in Buenos Aires, Argentina

17. Children with HUS at the Hospital de Niños, Dr. Ricardo Gutierrez, Buenos Aires, Argentina Age (months)

18. Origin of Shiga toxin-producing Escherichia coli strains Country Serotype Number of Origin Time of Strains Isolation Argentina Non-O157 8 Healthy steers 1997-1998 7 Healthy calves 1999-2000 O157:H72 Healthy and diarrheic 1998-1999 Veal Holstein calves Non-O157 9 Frozen Hamburgers 1998-2000 1 soft cheese sample O157:H7 6 Ground beef sample 2000 Non-O157 1 O157:H7 1 0 Animal and meat sample 1996-2000 Brazil O157:H7 3 Healthy calves, heifers 1996-1997 and cows Non-O157 4 O157:NM 1 Calves with diarrhea Unknown Non-O157 3 Non-O157 9 Ground beef and 1995-1996 hamburger sample

19. Enterohemorrhagic E. coli O157:H7 in Latin American Countries

20. In Mexico, there have been no reports of HC or HUS associated with O157:H7 strains However, studies from our laboratory conducted between 1985 and 2003, isolated O157 non-motile or non-H7 strains in approx. 10% of the children studied

21. Escherichia coli O157 strains isolated in Mexico 1985-2003 N= 263

22. The responses of 605 human serum samples against O157 LPS and its two cross-reacting LPSs, O7 and O116, were analyzed by ELISA. Of the 605 samples, 562 came from adolescents or adults and 43 from children of different age groups.

23. Human serum responses against different E. coli LPS’s by ELISA test using a cut-off point of 0.7 Serum samples E. coli LPS n(%) O7 (%) O116 (%) O157 (%) Children 43 1 (2) 5 (12) 2 (5) Adults 562 7 (1) 27 (5) 28 (5) Total 605 8 (1) 32 (5) 30 (5)

24. Comparative responses of sera from children against different E. coli LPS’s determined by ELISA test using a cut-off point of >0.4-0.69 and >0.7 E. coli LPS (%)

25. Comparative responses of sera from adults against different E. coli LPS’s determined by ELISA test using a cut-off point of >0.4-0.69 and >0.7 E. coli LPS (%)

26. Immune response of human serum samples against E. coli O157 LPS by Western blotting using E. coli O157 LPS developed with rabbit and human serum samples. kDa

27. Immune response of human breast milk samples against E. coli O157 LPS kDa

28. Western blotting analysis of the serum samples with a positive ELISA result (using a cutoff point of >0.7) showed that 86% (24 of 28) reacted with the O157 LPS. Further studies with breast milk and bovine serum samples showed that 71% and 23% respectively had a similar LPS response to that found in human serum studies.

29. * PCR Escherichia coli O157 isolated from Animals in Mexico from 1995-1996 N= 132

30. Heterologous response of bovine serum samples against different LPS’s of E. coli N= 310 Serum dilutions Positive samples

31. Response of bovine serum samples against O157 and cross-reacting LPS’s LPS N=310 ELISA cut-off: 0.7 DO a 405nm. Serum dilution: 1:800

32. These results suggest that early colonization by a non-pathogenic E. coli with an LPS that cross-reacts with O157 could be related to a low incidence of HC or HUS in children or adults from developing countries.