1. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Study Design and Measures of Association Dr Danilo Lo Fo Wong

2. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Outline Study objectives Types of study design Measures of association

3. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Study objectives Investigate an outbreak Measure disease frequency Assess risk factors Investigate trends Hypothesis generating Hypothesis testing

4. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Main types of studies Experimental studies Observational studies Cross-sectional studies Longitudinal studies Case-control studies Cohort studies Clinical trials

5. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Observational studies Evaluate the association between exposure (E) and disease outcome (D) No random allocation of exposure or intervention Harmonise groups as much as possible except for the exposures under investigation

6. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Observational studies Time D+ D-DiseasedNon-diseased E+ E-ExposedNon-exposed Study onset

7. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Cohort study (prospective) E+ E- D+ D- D+ D- Time

8. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Cohort study (retrospective) E+ E- D+ D- D+ D- Time

9. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Cohort study Applications: (Foodborne) disease outbreak Usually high attack rates Identifiable & manageable setting –Single event (wedding, picnic, etc.) –Single location (cafeteria, restaurant, etc.) Assess (foodborne) disease incidence Assess risk factors

10. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Cohort study Advantages Clear temporal relationship Least susceptible to some forms of bias Possibility to examine multiple diseases Disadvantages No control over exposure Not suitable for rare or long- latent diseases Loss to follow-up threatens validity More expensive than other observational designs

11. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Case-control study D+ D- E+ E- E+ E- Time

12. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Case-control study Applications: (Foodborne) disease outbreak Unclear setting of exposure –Spread out over time and space –Large cohort and/or rare disease Increase in reported cases Assess risk factors for sporadic illness

13. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Case-control study Advantages Efficient use of time and resources Well-suited for rare diseases Examine multiple exposures Relatively inexpensive Best when cohort study is not feasible Disadvantages Not suited for rare exposures Difficult to identify appropriate controls Ascertaining historic exposure often difficult Sensitive to some forms of bias

14. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Cross-sectional study Time D+ D- E+ E-

15. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Cross-sectional study Applications: Prevalence estimate Screening Risk factor studies Hypothesis generating

16. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Cross-sectional study Advantages One stop, one time Least expensive Based on sample of general population –not restricted to people seeking medical care Disadvantages Not suitable if disease is rare or of short duration Not possible to determine temporal relationship

17. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Steps to develop a study Hypothesis generation Develop study objectives Decide which type of study works best Study methods –Design survey tool/questionnaire –Identify subjects ((non)diseased or (non)exposed) –Determine sample size

18. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Steps to develop a study Data gathering –Actively acquired data –Registered data –Published data Statistical analysis –Descriptive –Analytic Univariate Multivariate

19. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 2 x 2 Table a. No. of subjects that are exposed and have the disease b. No. of subjects that are exposed and do not have the disease c. No. of subjects that are not exposed and have the disease d. No. of subjects that are not exposed and do not have the disease ab cd NoYesExposure YesNoDisease a+bc+d a+c b+dn

20. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Measures of association Disease frequency (absolute) –Attack rate Strength of association (relative) –Relative risk (RR) –Odds ratio (OR) Significance of association –Chi-squared –P-values –Confidence intervals (CI)

21. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Disease frequency Attack rate: –the proportion of subjects at risk that develop disease (i.e. proportion of exposed subjects that become ill)

22. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 2 x 2 Table ab cd Yes No Yes No Exposure Disease Attack rate exposed : (diseased|exposed)/exposed = a/(a+b) a+b c+d a+cb+dn Attack rate unexposed : (diseased|unexposed)/unexposed = c/(c+d)

23. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Example 40400 20420 Yes No Yes No Eating product X Salmonellosis 440 440 60820880 Attack rate exposed : 40/440 = 0.09 = 9% Attack rate unexposed : 20/440 = 0.05 = 5%

24. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Strength of association Relative Risk (RR) –Risk of disease for exposed vs. non-exposed –Cohort and cross-sectional studies Odds ratio (OR) –Odds of exposure for diseased vs. non-diseased –Case-control, cross-sectional and cohort studies OR ~ RR when: –Cases and controls representative for population –Disease not frequent

25. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 2 x 2 Table ab cd Yes No Yes No Exposure Disease a+b c+d a+cb+dn OR = odds exposure diseased /odds exposure non-diseased = (a:c)/(b:d) = ad/bc RR = Risk exposed /Risk unexposed = [a/a+b)]/[c/c+d)]

26. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Example 40400 20420 Yes No Yes No Eating product X Salmonellosis 440 440 60820880 OR = (40*420)/(20*400) = 2.1 RR = (40/440)/(20/440) = 2.0

27. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Significance of association Chi-squared –Test association between exposure and disease P-value –probability that an association at least as strong as that observed might have arisen by chance alone –P < 0.05: significant Confidence interval (CI) –range within which in 95% of times the true value of the estimated association lies (i.e. 95% CI) –Indication of confidence in the point-estimate

28. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 2 x 2 Table ab cd Yes No Yes No Exposure Disease a+b c+d a+cb+dn Chi-squared: (a+b)(c+d)(a+c)(b+d) n(|ad-bc| - n/2) 2 P-value = Percentiles of Chi-square distribution

29. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Example 40400 20420 Yes No Yes No Eating product X Salmonellosis 440 440 60820880 Chi-squared: 7.15 P-value < 0.05

30. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Confidence Interval Lower border = ln(OR) – Z*(var ln(OR)) ½ = L 95% Confidence Interval for OR: e L to e U = X to Y Upper border = ln(OR) + Z*(var ln(OR)) ½ = U ab cd Yes No Yes No Exposure Disease a+b c+d a+cb+dn

31. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Example 40400 20420 Yes No Yes No Eating product X Salmonellosis 440 440 60820880 Lower border = 0.74 – 1.96*(0.08) ½ = 0.2 95% Confidence Interval for OR: e 0.2 to e 1.3 = 1.2 to 3.7 Upper border = 0.74 + 1.96*(0.08) ½ = 1.3

32. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Interpretation RR and CI RR > 1 : risk of disease among exposed greater than among unexposed RR = 1 : risk of disease the same among exposed and unexposed RR < 1 : risk of disease among exposed less than among unexposed (i.e. protective) CI lower < 1 < CI upper : no significant association

33. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Interpretation OR and CI OR > 1 : odds of exposure among diseased greater than among non-diseased OR = 1 : odds of exposure the same among diseased and non-diseased OR < 1 : odds of exposure among diseased less than among non-diseased (i.e. protective) CI lower < 1 < CI upper : no significant association

34. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Examples OR = 2.3; 95% CI 1.2 – 28.5 OR = 2.3; 95% CI 1.2 – 28.5 RR = 3.6; 95% CI 0.8 – 4.7 RR = 3.6; 95% CI 0.8 – 4.7 OR = 0.4; 95% CI 0.2 – 0.7 OR = 0.4; 95% CI 0.2 – 0.7 RR = 5.6; 95% CI 5.3 – 5.9 RR = 5.6; 95% CI 5.3 – 5.9

35. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Epi Info

36. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Hypothesis testing Which study to design? Nipah virus infection among abattoir workers across Malaysia Cholera outbreak in Manila, Philippines Foodborne salmonellosis in the Institute of Health Sciences hostel, Muscat, Oman

37. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Nipah virus infection: Cross-sectional study Objective: To assess the prevalence of Nipah infection among abattoir workers and the association with pigs Serum sampling of workers from registered abattoirs in 11 of 13 states in Malaysia Exposure: pigs Outcome: antibodies (ab’s) against Nipah virus Abattoirs (n=143) Workers (n=435) –7 out of 435 infected (1.6%) slaughtering pigs –0 out of 233 infected (0%) slaughtered ruminants

38. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Nipah virus infection: Cross-sectional study 7 workers carrying ab’s against Nipah virus from abattoirs in 3 states with clinical outbreak among pig farmers Workers in these 3 states more likely to have Nipah antibody than those in other states –7/144 (4.9%) vs. 0/291 (0%), P < 0.001 Conclusion: –Nipah infection not widespread among abattoir workers in Malaysia –Infection linked to exposure to pigs

39. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Vibrio cholerae: Case-control study Objective: to investigate an increasing number of diarrhea cases from a district in Manila List of diarrhea cases from 3 hospitals in 1 district 338 suspect cholera cases admitted Rectal swabs, environmental survey, water samples Spot map to identify extent of outbreak Case control study in 4 areas with highest AR’s –56 cases –56 controls –Matched for age and gender

40. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Vibrio cholerae: Case-control study 2 protective factors –Boiling water prior to drinking (OR = 0.08, 95% CI 0.02 – 0.4) –Washing drinking water containers with soap and water (OR = 0.12, 95% CI 0.02 – 0.63) Inspection showed leaking water pipes, numerous illegal connections and unsanitary toilet facilities –1 sample from household faucets positive for fecal coliforms Conclusion: Cholera outbreak due to contaminated water

41. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Salmonellosis Outbreak: Cohort study Questionnaire to all students at IOHS –123 cases identified among 158 who ate dinner (AR = 78%); 72 confirmed cases (S. Enteritidis) –0 cases out of 91 who did not eat this meal (AR = 0%) –RR infinity, p < 0.001 Univariate analysis –Chicken (RR = 4.4; 95% CI 1.7 – 11) –Egg mousse (RR = 6.0; 95% CI 2.5 – 15) –Fried dal (RR = 1.2; 95% CI 1.0 – 1.4) –Omelet (RR = 1.3; 95% CI 1.2 – 1.4) –Chapati (RR = 6.0; 95% CI 1.7 – 11)

42. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Salmonellosis Outbreak: Cohort study Restricted analysis (n=158) –Chicken (RR = 14; 95% CI 2.0 – 96) –Egg mousse (RR = 19; 95% CI 2.8 – 128) –Chapati (RR = 19; 95% CI 2.8 – 128) Interview of kitchen staff –Time- and temperature abuse of chicken –Cross-contamination of chapati dough with raw eggs –Mousse contained raw eggs and was not cooked Conclusion: Outbreak due to cross-contamination and time- temperature abuse during preparation of three foods in the IOHS kitchen

43. Foodborne Outbreak Investigation, Hanoi, Vietnam 01 – 05 June 2009 Thank you for your attention