1. Monitoring of Vibrio cholerae in Environmental Reservoirs in Haiti
Meer Taifur Alam
Emerging Pathogens Institute
University of Florida

2. Introduction
Cholera appeared in epidemic form in Haiti in 2010 for the first time in over 100 years and has continued to occur in subsequent years
As of January 2014, about 698,000 cases and more than 8,500 deaths have been reported
It is essential to understand patterns of ongoing cholera transmission and seasonality of cholera in Haiti and to assess the likelihood of future epidemics
This raises the question of whether the microorganism has established in environmental reservoirs in Haiti
If so, which environmental factors enhance the survival of the bacterium?

3. Objectives Selection of environmental sites to monitor V. cholerae
Observation of the physical parameters of environmental reservoirs and their correlation to the occurrence of V. cholerae
Isolation of V. cholerae from environmental reservoirs
Genetic characterization of the isolated V. cholerae

4. Sample Collection Sites
Fifteen fixed environmental sites along the transects of 3 rivers in the Gressier/ Leogane region including one independent estuarine site
Momance River (4 up-river sites and one estuarine site)
Gressier River (4 up-river sites and one estuarine site)
Tapion River (4 river sites)
Four-a-chaux (an independent estuarine site)
All sites were at least a half-mile apart, with the exception of the Christianville bridge and spring sites, which were 0.25 miles apart
Samples were collected on a monthly basis from each site between April, 2012 and March 2013

5. Sample Collection Sites

6. Physical Parameters Temperature pH Dissolved oxygen (DO)
Total dissolved solids (TDS)
Conductivity
Salinity

7. Physical Parameters Results
Conditional logistic regression with water quality factors and V. cholerae O1
Water quality
Units
Obs
Odds ratio
P value
[95% Conf. int.]
Temperature
1 °C 47
2.14
0.033*
1.06
4.31 pH
1 log[H+]
0.01
0.083
0.00
1.81
Dissolved oxygen
1 mg/L
0.32
0.091
0.08
1.20
Total dissolved solids
100 mg/L
1.08
0.258
0.95
1.23
Salinity
1 g/L
1.24
0.254
0.86
1.80
Conductivity
100 (µS/cm)
1.05
0.198
0.98
1.13
Heterotrophic bacteria
log (CFU/ml)
6.00
0.135
0.57
62.78
Conditional logistic regression with water quality factors and V. cholerae Non-O1
154
1.36
0.02*
1.76
0.44
0.311
0.09
0.50
0.003*
0.79
0.96
0.413
1.19
0.378
0.80
1.77
0.432
0.92
1.04
153
2.35
0.063
5.77

8. Correlation of V. cholerae with Temperature

9. Bacteriological Assays
Conventional culture and isolation of V. cholerae
Serology to confirm V. cholerae O1
PCR assay to identify Vibrio specific genes
Aerobic plate count

10. Isolation of V. cholerae from Environment April 2012-March 2013
Isolates
Environmental Samples (n=176)
No. positive
% positive
V. cholerae O1 6
3.4
V. cholerae nonO1 /O139 50
27.9
Total 56
31.3

11. Distribution of V. cholerae O1

12. Distribution of V. cholerae non-O1/non-O139

13. PCR Analysis of V. cholerae Genes
Strain
PCR
MAMA-PCR
ompW
toxR
tcpACL
tcpAET
ctxA
ctxB
rstRET
rstRCL
rstCET
rstCCL
ctxBCL
ctxBET
Env-9 + -
Env- 90
Env -94
Env -122
Env-383
Env-390
Env-114

14. PCR Analysis of V. cholerae Genes
Strain
PCR
MAMA-PCR
ompW
toxR
tcpACL
tcpAET
ctxA
ctxB
rstRET
rstRCL
rstCET
rstCCL
ctxBCL
ctxBET
Env-9 + -
Env- 90
Env -94
Env -122
Env-383
Env-390
**Env-114
** A shrimp isolate

15. Conclusions
Vibrio cholerae O1 and non-O1 were successfully isolated from the environment in Haiti
Isolation of non-O1 strains in environmental reservoirs suggests that conditions are appropriate for establishment of O1 strains at these same sites
Novel Vibrio cholerae O1 type biotype El Tor strain was isolated which lost its entire ctx phage
Water temperature emerged as statistically significant in a conditional logistic regression model

16. Future Goals
Development of new molecular beacon hybridization probes for the identification of V. cholerae
Perform a more sensitive colony blot assay and correlate the results with culture data and physical parameters
Develop a new enrichment technique for V. cholerae isolation
Introduce membrane fecal coliform counts (mFC) to observe correlation between mFC and V. cholerae

17. Acknowledgments
Afsar Ali, PhD Valery Madsen De Beau Rochars, MD J. Glenn Morris Jr., PhD (EPI Director ) Christianville Foundation Haitian laboratory personnel (UF-EPI Haiti Lab 1-Gressier) This work is supported by: NIH grant RO1 AI (Drs. Morris and Ali) NIH grant RO1 AI3929 (Drs. R. Bradley Sack, John Hopkins University and Ali) DoD grant C0654_12_UN (Dr. Ali)

18. THANK YOU