Monitoring of Vibrio cholerae in Environmental Reservoirs in Haiti

Meer Taifur Alam Emerging Pathogens Institute University of Florida

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?

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

Sample Collection Sites

• Fifteen fixed environmental sites along the transects of 3 rivers in the Gressier/ Leogane region including one independent estuarine site

I. Momance River (4 up-river sites and one estuarine site)

II. Gressier River (4 up-river sites and one estuarine site)

III. Tapion River (4 river sites)

IV. 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

Sample Collection Sites

Physical Parameters

• Temperature

• pH

• Dissolved oxygen (DO)

• Total dissolved solids (TDS)

• Conductivity

• Salinity

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+] 47 0.01 0.083 0.00 1.81

Dissolved oxygen 1 mg/L 47 0.32 0.091 0.08 1.20

Total dissolved solids 100 mg/L 47 1.08 0.258 0.95 1.23

Salinity 1 g/L 47 1.24 0.254 0.86 1.80

Conductivity 100 (µS/cm) 47 1.05 0.198 0.98 1.13

Heterotrophic bacteria log (CFU/ml) 47 6.00 0.135 0.57 62.78

Conditional logistic regression with water quality factors and V. cholerae Non-O1

Water quality Units Obs Odds ratio P value [95% Conf. int.]

Temperature 1 °C 154 1.36 0.02* 1.05 1.76

pH 1 log[H+] 154 0.44 0.311 0.09 2.14

Dissolved oxygen 1 mg/L 154 0.50 0.003* 0.32 0.79

Total dissolved solids 100 mg/L 154 0.96 0.413 0.86 1.06

Salinity 1 g/L 154 1.19 0.378 0.80 1.77

Conductivity 100 (µS/cm) 154 0.98 0.432 0.92 1.04

Heterotrophic bacteria log (CFU/ml) 153 2.35 0.063 0.95 5.77

Physical Parameters Results

Correlation of V. cholerae with Temperature

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

Isolation of V. cholerae from EnvironmentApril 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

Distribution of V. cholerae O1

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

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 + + - + + + + - - - + -

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 + + - + + + + - + - + -

PCR Analysis of V. cholerae Genes

** A shrimp isolate

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

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

Afsar Ali, PhDValery Madsen De Beau Rochars, MDJ. Glenn Morris Jr., PhD (EPI Director )Christianville FoundationHaitian laboratory personnel (UF-EPI Haiti Lab 1-Gressier)

This work is supported by: NIH grant RO1 AI097405 (Drs. Morris and Ali) NIH grant RO1 AI3929 (Drs. R. Bradley Sack, John Hopkins University and Ali)DoD grant C0654_12_UN (Dr. Ali)

Acknowledgments

THANK YOU