1. EnterococciEnterococci but notbut not E.coliE.coli counts incounts in
drinking water are positively associateddrinking water are positively associated
with gastrointestinal disease risk in urbanwith gastrointestinal disease risk in urban
ZambiaZambia
UNC water and Health Conference 2016UNC water and Health Conference 2016
1313thth
October, 2016October, 2016
Michelo SimuyandiMichelo Simuyandi
PhD candidate: LSHTM-ITD-DCDPhD candidate: LSHTM-ITD-DCD
Research Fellow: CIDRZResearch Fellow: CIDRZ

2. Outline
• Background
• Methods
• Results
• Discussion
• Conclusion

3. Background
• Studies on the association between faecal indicator bacteria in drinking
water and diarrhoeal disease risk have yielded mixed findings that range
from no association to significant associations1,2
• This variation could be due to:
– high variability of water quality over time3
– Indicator organisms being weakly correlated to enteric pathogens4
– Study design related: collecting water quality and health data at the same
time may fail to determine direction of causality 5
– Acquired immunity to certain diarrhoea causing pathogens could affect
generalisation of the associations found in some studies6
– Other transmission routes of infection7
1. Gundry et al 2004
2. Gruber et al, 2014
3. Levy et al., 2009
4. Wu et al., 2011,
5. Khush et al., 2013
6. Frost et al., 2005
7. Luby et al 2015

4. Study design
• We conducted a prospective study collecting
repeated measures of water quality and
health indicators and related covariates at
both household and individual level in a peri-
urban area south of Lusaka
– As part of the pilot study of a field trial of a point
of use HWTS intervention

5. Aim
• To determine the association between the
levels of Enterococci, E. coli and total coliforms
in household drinking water and self-reported
highly credible gastrointestinal illness (HCGI)
experienced by household members using a 7
day recall period.

6. Definitions
• The exposure was defined as
– Safe = <1 MPN/100 mL
– Unsafe >1 MPN/100 mL
• Outcome measure used was self-reported highly credible
gastrointestinal illness (HCGI) experienced by household
members using a 7 day recall period
– HCGI : defined as a report of at least one of: 1) vomiting; 2) watery
diarrhoea; 3) soft diarrhoea and abdominal cramps both occurring
during recall period; 4 ) nausea and abdominal cramps both occurring
during recall period (Colford et al., 2005)
• The associations between Enterococci, E. coli, total coliforms
in household drinking water and the HCGI experienced by
household members were investigated using mixed-effects
logistic regression

7. Methods
• Data was collect by
– household interviews using a survey,
– household observations
• Testing of household stored drinking water
and source water for bacterial indicators of
faecal contamination was done using the
IDEXX quanti-tray, MPN Methods
– Enterolert® Test for Enterococci
– Colilert® Test for E coli and total coliforms

8. FACTS/stats
• Intermittent water supply
• unsafe sanitation
• high fertility
• High Unemployment rate
• poor drainage

10. 5 CEA Household
clusters(1206 HH)
300 households
recruited (60/cluster)
244 households included in
analysis
244 households
contributing to analysis
832 Individuals
466 females
196 children below 5yrs
10 households lost to follow-up
•8 relocated to area outside study
area.
•2 withdrew without reason
44 households excluded due to
missing/incomplete data
Inclusion criteria
At least a child below 5yrs, written
consent, purposive sampling till
agreed 60 hh /cluster

11. Household Characteristic Number of subjects Proportion(%)
(% of total) with HCGI (95% CI)1
Water Quality
Ecoli count (n=635)
<1 268(42.2) 42/339 (12.4) (8.8, 17.1)
1-10 127(20.0) 15/180 (8.3) (4.1, 16.3)
11-100 98(15.4) 13/131 (9.9) (4.8, 19.4)
101 + 142(22.4) 19/185 (10.3) (5.9, 17.2)
Total coliforms (Ecoli-media) (n=635)
<1 81(12.8) 12/98 (12.2) (6.6, 21.5)
1-10 48(7.6) 12/65 (18.5) (8.2, 36.5)
11-100 18(2.8) 2/40 (5.0) (1.3, 17.7)
101 + 488(76.9) 63/632 (10.0) (7.4, 13.3)
Enterococci count (n=570)
<1 529(92.8) 51/676 (7.5) (5.3, 10.6)
1-10 23(4.0) 5/23 (21.7) (6.2, 5.4)
11-100 12(2.1) 3/12 (25.0) (9.1, 52.6)
101 + 6(1.1) 2/7 (28.6) (8.2, 64.3)
Household Water Treatment (n=829)
No 661 (79.73) 89/692 (12.86) (9.92, 16,51)
Yes 168 (20.27) 21/280 (6.98) (4.45, 10.77)
Primary Water Source (n=569)
Tap inside house 53 (9.31) 5/67 (7.46) (3.94, 13.70)
Stand pipe 244 (42.88) 52/371 (14.02) (10.12, 19.09)
Other 272 (47.80) 52/406 (12.81) (9.17, 17.60)
Containers per daya
mean(SD) 3.16 (2.00)
Containers per day category (n=766)
0-2 347 (45.30) 40/472 (8.47) (5.62, 12.59)
3-5 307 (40.08) 62/468 (13.25) (9.86, 17.58)
6-9 112 (14.62) 22/181 (12.15) (7.86, 18.33)
Distance to water source (n=791)
<200 meters 762 (96.33) 128/1101 (11.63) (9.42, 14.26)
200+ metres 29 (3.67) 1/47 (2.13) (0.26, 15.25)
How long to collect water (n=371)
5-14 minutes 306 (82.48) 40/468 (8.55) (5.74, 12.55)
15+ minutes 65 (17.52) 11/85(12.94) (7.70, 20.99)
Household and individual baseline characteristics for the study population

12. Household Characteristic Number of subjects Proportion(%)
(% of total) with HCGI (95% CI)1
How long to collect water (n=371)
5-14 minutes 306 (82.48) 40/468 (8.55) (5.74, 12.55)
15+ minutes 65 (17.52) 11/85(12.94) (7.70, 20.99)
Safe Sanitationb
(n=695)
No 622 (89.50) 107/920 (11.63) (9.23, 14.55)
Yes 73 (10.5) 11/112 (9.82) (4.50, 20.11)
Good Hygienec
(n=821)
No 622 (75.76) 102/900 (11.33) (8.91, 14.30)
Yes 199 (24.24) 29/290 (10.00) (6.56, 14.96)
Place to Wash hands in house (n=827)
No 640 (77.39) 95/934 (10.17) (8.05, 12.78)
Yes 187 (22.61) 37/262 (14.12) (9.15, 21.16)
Place to Wash hands near toilet (n=829)
No 721 (86.97) 106/1038 (10.21) (8.10, 12.80)
Yes 108 (13.03) 26/160 (16.25) (10.00, 25.31)
Seasone
Wet 86/928(9.27) (7.21, 11.83)
Dry 46/273(16.85) (11.54,23.94)
Zone (n=832)
Zone A 113 (13.58) 9/145(6.21) (2.67, 13.76)
Zone B 172 (20.67) 41/229(17.90) (13.03, 24.09)
Zone C 249 (29.93) 41/366(11.20) (7.49, 16.44)
Zone D 176 (21.15) 17/254(6.69) (4.07, 10.82)
Zone E 122 (14.66) 24/207(11.59) (7.22, 18.10)
Individual Characteristics
Sex (n=824)
Female 358 (43.45) 82/677(12.11) (9.56, 15.23)
Male 466 (56.55) 48/510(9.41) (7.09, 12.40)
Age Mean(SD) 19.47 (16.35)
Age Category (n=825)
<5 years 196 (23.76) 37/268 (13.81) (10.03, 18.70)
>= 5 years 629 (76.24) 95/925 (10.27) (8.10, 12.92)
Total 832 (100%) 132/1201 (10.99) (8.93, 13.45)

13. Water quality:
A=Source vs HH stored and B =Storage container type

14. VariableVariable Number of Obs.Number of Obs.
(N=1,201) Odds Ratio 95% CI P-value
Age1
< 5 years 1,903 1.52 (0.95,2.44) 0.08
Sex (female) 1,187 1.40 (0.92,2.11) 0.12
Water quality
Unsafe E. coli count 270/835 0.59 (0.33,1.06) 0.08
Unsafe total coliforms 270/835 0.79 (0.35,1.79) 0.57
Unsafe Enterococci count 250/718 15.25 (2.27,102.27) 0.01
Treat Water1
993 0.41 (0.22,0.75) <0.01
Containers collected per day2
1,121 1.09 (0.96, 1.25) 0.19
Distance to Source > 200 meters2
1,148 0.16 (0.02,1.51) 0.11
>14 minutes to collect water2
553 1.85 (0.66, 5.14) 0.24
Narrow mouth container1
1,023 0.98 (0.53,1.84) 0.96
Good Hygiene2
1,190 0.85 (0.47, 1.54) 0.60
Place to wash hands in house2
1,196 1.46 (0.83, 2.60) 0.20
Place to wash hands near toilet2
1,198 2.12 (1.08, 4.17) 0.03
Safe Sanitation2
1,032 1.03 (0.40, 2.64) 0.95
Containers collected per day= number of 20 litre containers of water collected per day.
Good Hygiene=Have soap (shown to interviewer) and household respondent asserts washes with soap. Safe sanitation=Household respondent asserts that the
household uses a private latrine.
1
As reported at visit corresponding to HCGI outcome. (i.e. “Did you treat this water you are currently drinking?”)
2
As reported at baseline (first available visit for which response was available).
Mixed effects logistic regression accounting for household and individual-level clustering. All associations with p-value < 0.05 are underlined
Univariate associations with Highly Credible Gastrointestinal Illness

15. (Unadjusted) Adjusted
Variable n Odds Ratio 95%CI P-value Odds Ratio 95%CI P-value
Unsafe Enterococci count 528 26.55 (1.45, 486.04) 0.03 31.33 (2.13, 461.73) 0.01
Age < 5 years 528 2.36 (0.95, 5.84) 0.06 2.92 (0.85, 10.03) 0.09
Treat Water 528 0.41 (0.14, 1.22) 0.11
Place to wash hands near
toilet
528 1.87 (0.46, 7.52) 0.38
Mixed effects logistic regression (accounting for household and individual-level clustering) using a
backwards selection procedure; nested models were evaluated using likelihood ratio tests with a p<0.1
threshold.
‘Treat water’ and ‘Place to wash hands near toilet’ were dropped from final model. P-values <0.05 are
underlined.
Mixed effects logistic regression accounting for household and
individual-level clustering

16. Discussion
• Within the limitations of the study design, data and analysis
– Enterococci counts and not Ecoli and total coliforms is potentially
associated with HCGI.
• Risebro et al (2012) reported a strong association between Enterococci counts and
infectious intestinal disease as opposed to E.coli counts in England
– Reported household treatment of drinking water was negatively
associated with HCGI only in the unadjusted analysis 0.41 (0.22,0.75)
<0.01
– Place to wash hands near toilet was positively associated with HCGI
only in the unadjusted analysis (OR 2.12 (1.08, 4.17)

17. Conclusion
• Given higher percentage of samples that were
unsafe (E.coli and total coliforms) makes these
potentially poor predictors (i.e., there is E. coli
everywhere!
• In this urban setting, Enterococci counts have
a potential association with HCGI than E.coli
or total coliform counts.
• Need to validate Enterococci counts in this
and other settings as stronger indicator for
risk of HCGI

18. Acknowledgements
• Funding
– Research funding NIH-through my supervisor: Joe Brown
– Travel scholarship- BMGF through UNC Water & Health
Institute
• Tropical Gastroenterology group
– Research host site/study research team
• LSHTM-EHG
– Host research group for my PhD
• Centre for Infectious Disease Research in Zambia
– Enteric Disease and Vaccines Research Unit
• Employers, local PhD advisory team
• Misisi community
– Study participants

19. Comments and Questions?