ABSTRACTCholera is an acute watery diarrheal disease that causes high rates of morbidity and mort... more ABSTRACTCholera is an acute watery diarrheal disease that causes high rates of morbidity and mortality without treatment. Early detection of the etiologic agent of toxigenic Vibrio cholerae is important to mobilize treatment and mitigate outbreaks. Monoclonal antibody (mAb) based rapid diagnostic tests (RDTs) enable early detection in settings without laboratory capacity. However, the odds of an RDT testing positive are reduced by nearly 90% when the common virulent bacteriophage ICP1 is present. We hypothesize that adding a mAb for the common, and specific, virulent bacteriophage ICP1 as a proxy for V. cholerae to an RDT will increase diagnostic sensitivity when virulent ICP1 phage are present. In this study, we used an in-silico approach to identify immunogenic ICP1 protein targets that were conserved across disparate time periods and locations. Specificity of targets to cholera patients with known ICP1 was determined, and specific targets were used to produce mAbs in a murine mod...
Bacteriophage predation of toxigenic Vibrio cholerae O1 (the causative agent of cholera) has been... more Bacteriophage predation of toxigenic Vibrio cholerae O1 (the causative agent of cholera) has been linked with seasonal patterns of disease and with clinical response to infection in humans 1-4. However, we still lack a clear demonstration of how the interplay between bacteria and bacteriophage can influence shifts in strain populations. We analyzed toxigenic V. cholerae O1 isolated from patients in the Great Lakes, a cholera endemic region of the Democratic Republic of the Congo (DRC), between 2013-2017. Bayesian phylogeography shows that all strains derived from the East Africa T10 introduction event5, consistent with establishment of a regional endemic focus, and identified two major lineages, with the most recent correlating to ST515, a cholera cluster previously found in the Lake Kivu and expanding northward6. We also identified a novel ICP1 bacteriophage, genetically distinct from previous ICP1 isolates detected in Asia 7,8, from stool samples of cholera patients. The presence ...
In order to provide our readers with timely access to new content, papers accepted by the America... more In order to provide our readers with timely access to new content, papers accepted by the American Journal of Tropical Medicine and Hygiene are posted online ahead of print publication. Papers that have been accepted for publication are peer-reviewed and copy edited but do not incorporate all corrections or constitute the final versions that will appear in the Journal. Final, corrected papers will be published online concurrent with the release of the print issue. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Vibrio cholerae is a halophilic Gram-negative bacterial species and the etiological agent of chol... more Vibrio cholerae is a halophilic Gram-negative bacterial species and the etiological agent of cholera. Here, we report the draft genome sequence of an environmental V. cholerae strain, 2012Env-25, obtained using Oxford Nanopore Technologies (ONT) to provide insights into the ecology, evolution, and pathogenic potential of this bacterium.
Proceedings of the National Academy of Sciences, 2020
The spread of cholera in the midst of an epidemic is largely driven by direct transmission from p... more The spread of cholera in the midst of an epidemic is largely driven by direct transmission from person to person, although it is well-recognized thatVibrio choleraeis also capable of growth and long-term survival in aquatic ecosystems. While prior studies have shown that aquatic reservoirs are important in the persistence of the disease on the Indian subcontinent, an epidemiological view postulating that locally evolving environmentalV. choleraecontributes to outbreaks outside Asia remains debated. The single-source introduction of toxigenicV. choleraeO1 in Haiti, one of the largest outbreaks occurring this century, with 812,586 suspected cases and 9,606 deaths reported through July 2018, provided a unique opportunity to evaluate the role of aquatic reservoirs and assess bacterial transmission dynamics across environmental boundaries. To this end, we investigated the phylogeography of both clinical and aquatic toxigenicV. choleraeO1 isolates and show robust evidence of the establish...
International Journal of Environmental Research and Public Health, 2019
Diffusion of cholera and other diarrheal diseases in an informal settlement is a product of multi... more Diffusion of cholera and other diarrheal diseases in an informal settlement is a product of multiple behavioral, environmental and spatial risk factors. One of the most important components is the spatial interconnections among water points, drainage ditches, toilets and the intervening environment. This risk is also longitudinal and variable as water points fluctuate in relation to bacterial contamination. In this paper we consider part of this micro space complexity for three informal settlements in Port au Prince, Haiti. We expand on more typical epidemiological analysis of fecal coliforms at water points, drainage ditches and ocean sites by considering the importance of single point location fluctuation coupled with recording micro-space environmental conditions around each sample site. Results show that spatial variation in enteric disease risk occurs within neighborhoods, and that while certain trends are evident, the degree of individual site fluctuation should question the u...
During the cholera outbreak from 2010 to 2011 in Cameroon, 33,192 cases with 1,440 deaths (case f... more During the cholera outbreak from 2010 to 2011 in Cameroon, 33,192 cases with 1,440 deaths (case fatality ratio 4.34%) were reported to the World Health Organization. Of these, the South West Region reported 3,120 clinical cases. This region is in the Equatorial Monsoon climatic subzone of Cameroon, close to the coast, raising questions as to whether cases were linked with development of environmental reservoirs. In an investigation conducted by the Laboratory for Emerging Infectious Diseases, University of Buea, toxigenic V. cholerae O1 were isolated from diarrheal stool samples from 18 patients, with ages ranging from <3 to 70 years. Coordinates for clinical centers at which cases were identified were obtained using a handheld GPS, and were mapped using ArcGIS. Antibiotic susceptibility testing was performed using the Kirby 'Bauer agar disc diffusion method. The full genomes of these strains were sequenced with the Illumina MiSeq platform. De novo assembly of cholera genomes...
International journal of environmental research and public health, Jan 3, 2016
The cartographic challenge in many developing world environments suffering a high disease burden ... more The cartographic challenge in many developing world environments suffering a high disease burden is a lack of granular environmental covariates suitable for modeling disease outcomes. As a result, epidemiological questions, such as how disease diffuses at intra urban scales are extremely difficult to answer. This paper presents a novel geospatial methodology, spatial video, which can be used to collect and map environmental covariates, while also supporting field epidemiology. An example of epidemic cholera in a coastal town of Haiti is used to illustrate the potential of this new method. Water risks from a 2012 spatial video collection are used to guide a 2014 survey, which concurrently included the collection of water samples, two of which resulted in positive lab results "of interest" (bacteriophage specific for clinical cholera strains) to the current cholera situation. By overlaying sample sites on 2012 water risk maps, a further fifteen proposed water sample location...
Shiga toxins (Stx) are commonly produced by Shigella dysenteriae serotype 1 and Stx-producing Esc... more Shiga toxins (Stx) are commonly produced by Shigella dysenteriae serotype 1 and Stx-producing Escherichia coli. However, the toxin genes have been detected in additional Shigella species. We recently reported the emergence of Stx-producing Shigella in travelers in the United States and France who had recently visited Hispaniola (Haiti and the Dominican Republic). In this study, we confirm this epidemiological link by identifying Stx-producing Shigella from Haitian patients attending clinics near Port-au-Prince. We also demonstrate that the bacteriophage encoding Stx is capable of dissemination to stx-negative Shigella species found in Haiti, suggesting that Stx-producing Shigella may become more widespread within that region.
Since the identification of the first cholera case in 2010, the disease has spread in epidemic fo... more Since the identification of the first cholera case in 2010, the disease has spread in epidemic form throughout the island nation of Haiti; as of 2014, about 700,000 cholera cases have been reported, with over 8,000 deaths. While case numbers have declined, the more fundamental question of whether the causative bacterium, Vibrio cholerae has established an environmental reservoir in the surface waters of Haiti remains to be elucidated. In a previous study conducted between April 2012 and March 2013, we reported the isolation of toxigenic V. cholerae O1 from surface waters in the Ouest Department. After a second year of surveillance (April 2013 to March 2014) using identical methodology, we observed a more than five-fold increase in the number of water samples containing culturable V. cholerae O1 compared to the previous year (1.7% vs 8.6%), with double the number of sites having at least one positive sample (58% vs 20%). Both seasonal water temperatures and precipitation were signifi...
The American journal of tropical medicine and hygiene, 2014
We inventoried non-surface water sources in the Leogane and Gressier region of Haiti (approximate... more We inventoried non-surface water sources in the Leogane and Gressier region of Haiti (approximately 270 km(2)) in 2012 and 2013 and screened water from 345 sites for fecal coliforms and Vibrio cholerae. An international organization/non-governmental organization responsible for construction could be identified for only 56% of water points evaluated. Sixteen percent of water points were non-functional at any given time; 37% had evidence of fecal contamination, with spatial clustering of contaminated sites. Among improved water sources (76% of sites), 24.6% had fecal coliforms versus 80.9% in unimproved sources. Fecal contamination levels increased significantly from 36% to 51% immediately after the passage of Tropical Storm Sandy in October of 2012, with a return to 34% contamination in March of 2013. Long-term sustainability of potable water delivery at a regional scale requires ongoing assessment of water quality, functionality, and development of community-based management schemes...
The 2010 earthquake in Haiti led to thousands of deaths, destruction of drinking and waste-water ... more The 2010 earthquake in Haiti led to thousands of deaths, destruction of drinking and waste-water infrastructure and displacement of millions into temporary camps with little sanitation and hygiene. Even worse, the introduction of Vibrio cholera in October, 2010 resulted in a massive cholera outbreak that spread rapidly throughout Haiti. Once it was recognized that many displaced persons lacked access to water and the outbreak likely resulted from consumption of contaminated surface water, thousands of wells were installed by Non-Government Organizations. However, despite an ongoing epidemic, no water quality data was collected from these wells to verify their safety. To determine if the wells were a source of exposure, 359 sources of drinking water in the Leogane flood basin, located at ground zero of the earthquake, were screened for V. cholerae and fecal coliform bacteria. While no toxigenic strains of V. cholerae were identified, non-toxigenic V. cholerae was isolated from six wa...
Phylodynamic analysis of genome-wide single-nucleotide polymorphism (SNP) data is a powerful tool... more Phylodynamic analysis of genome-wide single-nucleotide polymorphism (SNP) data is a powerful tool to investigate underlying evolutionary processes of bacterial epidemics. The method was applied to investigate a collection of 65 clinical and environmental isolates of Vibrio cholerae from Haiti collected between 2010 and 2012. Characterization of isolates recovered from environmental samples identified a total of four toxigenic V. cholerae O1 isolates, four non-O1/O139 isolates, and a novel nontoxigenic V. cholerae O1 isolate with the classical tcpA gene. Phylogenies of strains were inferred from genome-wide SNPs using coalescent-based demographic models within a Bayesian framework. A close phylogenetic relationship between clinical and environmental toxigenic V. cholerae O1 strains was observed. As cholera spread throughout Haiti between October 2010 and August 2012, the population size initially increased and then fluctuated over time. Selection analysis along internal branches of t...
Extracts in organic solvents (namely methanol, ethanol, ethyl acetate and chloroform) of two medi... more Extracts in organic solvents (namely methanol, ethanol, ethyl acetate and chloroform) of two medicinal plants - Achyranthes aspera and Cassia alata were evaluated for their antibacterial activities against Escherichia coli, Bacillus subtilis, Vibrio cholerae, Salmonella typhi and Staphylococcus aureus. These were carried out by taking the organic extracts of both the leaf and stem parts of the plants at a concentration of 5 mg/ml and their activities were recorded by estimating zones of inhibition as produced by disc-diffusion method on Mueller-Hinton agar media. While neither the leaf nor stem parts of A. aspera in any organic extractions showed antibacterial activity, the methanolic extracts of both the leaf and stem parts of C. alata exhibited antibacterial activity, but only to B. subtilis and S. typhi, and the corresponding MIC values of the leaf extracts were estimated as 1.25 and 1.5 mg/ml respectively. However, the ethanolic extracts of both the stem and leaf parts were foun...
ABSTRACTCholera is an acute watery diarrheal disease that causes high rates of morbidity and mort... more ABSTRACTCholera is an acute watery diarrheal disease that causes high rates of morbidity and mortality without treatment. Early detection of the etiologic agent of toxigenic Vibrio cholerae is important to mobilize treatment and mitigate outbreaks. Monoclonal antibody (mAb) based rapid diagnostic tests (RDTs) enable early detection in settings without laboratory capacity. However, the odds of an RDT testing positive are reduced by nearly 90% when the common virulent bacteriophage ICP1 is present. We hypothesize that adding a mAb for the common, and specific, virulent bacteriophage ICP1 as a proxy for V. cholerae to an RDT will increase diagnostic sensitivity when virulent ICP1 phage are present. In this study, we used an in-silico approach to identify immunogenic ICP1 protein targets that were conserved across disparate time periods and locations. Specificity of targets to cholera patients with known ICP1 was determined, and specific targets were used to produce mAbs in a murine mod...
Bacteriophage predation of toxigenic Vibrio cholerae O1 (the causative agent of cholera) has been... more Bacteriophage predation of toxigenic Vibrio cholerae O1 (the causative agent of cholera) has been linked with seasonal patterns of disease and with clinical response to infection in humans 1-4. However, we still lack a clear demonstration of how the interplay between bacteria and bacteriophage can influence shifts in strain populations. We analyzed toxigenic V. cholerae O1 isolated from patients in the Great Lakes, a cholera endemic region of the Democratic Republic of the Congo (DRC), between 2013-2017. Bayesian phylogeography shows that all strains derived from the East Africa T10 introduction event5, consistent with establishment of a regional endemic focus, and identified two major lineages, with the most recent correlating to ST515, a cholera cluster previously found in the Lake Kivu and expanding northward6. We also identified a novel ICP1 bacteriophage, genetically distinct from previous ICP1 isolates detected in Asia 7,8, from stool samples of cholera patients. The presence ...
In order to provide our readers with timely access to new content, papers accepted by the America... more In order to provide our readers with timely access to new content, papers accepted by the American Journal of Tropical Medicine and Hygiene are posted online ahead of print publication. Papers that have been accepted for publication are peer-reviewed and copy edited but do not incorporate all corrections or constitute the final versions that will appear in the Journal. Final, corrected papers will be published online concurrent with the release of the print issue. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Vibrio cholerae is a halophilic Gram-negative bacterial species and the etiological agent of chol... more Vibrio cholerae is a halophilic Gram-negative bacterial species and the etiological agent of cholera. Here, we report the draft genome sequence of an environmental V. cholerae strain, 2012Env-25, obtained using Oxford Nanopore Technologies (ONT) to provide insights into the ecology, evolution, and pathogenic potential of this bacterium.
Proceedings of the National Academy of Sciences, 2020
The spread of cholera in the midst of an epidemic is largely driven by direct transmission from p... more The spread of cholera in the midst of an epidemic is largely driven by direct transmission from person to person, although it is well-recognized thatVibrio choleraeis also capable of growth and long-term survival in aquatic ecosystems. While prior studies have shown that aquatic reservoirs are important in the persistence of the disease on the Indian subcontinent, an epidemiological view postulating that locally evolving environmentalV. choleraecontributes to outbreaks outside Asia remains debated. The single-source introduction of toxigenicV. choleraeO1 in Haiti, one of the largest outbreaks occurring this century, with 812,586 suspected cases and 9,606 deaths reported through July 2018, provided a unique opportunity to evaluate the role of aquatic reservoirs and assess bacterial transmission dynamics across environmental boundaries. To this end, we investigated the phylogeography of both clinical and aquatic toxigenicV. choleraeO1 isolates and show robust evidence of the establish...
International Journal of Environmental Research and Public Health, 2019
Diffusion of cholera and other diarrheal diseases in an informal settlement is a product of multi... more Diffusion of cholera and other diarrheal diseases in an informal settlement is a product of multiple behavioral, environmental and spatial risk factors. One of the most important components is the spatial interconnections among water points, drainage ditches, toilets and the intervening environment. This risk is also longitudinal and variable as water points fluctuate in relation to bacterial contamination. In this paper we consider part of this micro space complexity for three informal settlements in Port au Prince, Haiti. We expand on more typical epidemiological analysis of fecal coliforms at water points, drainage ditches and ocean sites by considering the importance of single point location fluctuation coupled with recording micro-space environmental conditions around each sample site. Results show that spatial variation in enteric disease risk occurs within neighborhoods, and that while certain trends are evident, the degree of individual site fluctuation should question the u...
During the cholera outbreak from 2010 to 2011 in Cameroon, 33,192 cases with 1,440 deaths (case f... more During the cholera outbreak from 2010 to 2011 in Cameroon, 33,192 cases with 1,440 deaths (case fatality ratio 4.34%) were reported to the World Health Organization. Of these, the South West Region reported 3,120 clinical cases. This region is in the Equatorial Monsoon climatic subzone of Cameroon, close to the coast, raising questions as to whether cases were linked with development of environmental reservoirs. In an investigation conducted by the Laboratory for Emerging Infectious Diseases, University of Buea, toxigenic V. cholerae O1 were isolated from diarrheal stool samples from 18 patients, with ages ranging from <3 to 70 years. Coordinates for clinical centers at which cases were identified were obtained using a handheld GPS, and were mapped using ArcGIS. Antibiotic susceptibility testing was performed using the Kirby 'Bauer agar disc diffusion method. The full genomes of these strains were sequenced with the Illumina MiSeq platform. De novo assembly of cholera genomes...
International journal of environmental research and public health, Jan 3, 2016
The cartographic challenge in many developing world environments suffering a high disease burden ... more The cartographic challenge in many developing world environments suffering a high disease burden is a lack of granular environmental covariates suitable for modeling disease outcomes. As a result, epidemiological questions, such as how disease diffuses at intra urban scales are extremely difficult to answer. This paper presents a novel geospatial methodology, spatial video, which can be used to collect and map environmental covariates, while also supporting field epidemiology. An example of epidemic cholera in a coastal town of Haiti is used to illustrate the potential of this new method. Water risks from a 2012 spatial video collection are used to guide a 2014 survey, which concurrently included the collection of water samples, two of which resulted in positive lab results "of interest" (bacteriophage specific for clinical cholera strains) to the current cholera situation. By overlaying sample sites on 2012 water risk maps, a further fifteen proposed water sample location...
Shiga toxins (Stx) are commonly produced by Shigella dysenteriae serotype 1 and Stx-producing Esc... more Shiga toxins (Stx) are commonly produced by Shigella dysenteriae serotype 1 and Stx-producing Escherichia coli. However, the toxin genes have been detected in additional Shigella species. We recently reported the emergence of Stx-producing Shigella in travelers in the United States and France who had recently visited Hispaniola (Haiti and the Dominican Republic). In this study, we confirm this epidemiological link by identifying Stx-producing Shigella from Haitian patients attending clinics near Port-au-Prince. We also demonstrate that the bacteriophage encoding Stx is capable of dissemination to stx-negative Shigella species found in Haiti, suggesting that Stx-producing Shigella may become more widespread within that region.
Since the identification of the first cholera case in 2010, the disease has spread in epidemic fo... more Since the identification of the first cholera case in 2010, the disease has spread in epidemic form throughout the island nation of Haiti; as of 2014, about 700,000 cholera cases have been reported, with over 8,000 deaths. While case numbers have declined, the more fundamental question of whether the causative bacterium, Vibrio cholerae has established an environmental reservoir in the surface waters of Haiti remains to be elucidated. In a previous study conducted between April 2012 and March 2013, we reported the isolation of toxigenic V. cholerae O1 from surface waters in the Ouest Department. After a second year of surveillance (April 2013 to March 2014) using identical methodology, we observed a more than five-fold increase in the number of water samples containing culturable V. cholerae O1 compared to the previous year (1.7% vs 8.6%), with double the number of sites having at least one positive sample (58% vs 20%). Both seasonal water temperatures and precipitation were signifi...
The American journal of tropical medicine and hygiene, 2014
We inventoried non-surface water sources in the Leogane and Gressier region of Haiti (approximate... more We inventoried non-surface water sources in the Leogane and Gressier region of Haiti (approximately 270 km(2)) in 2012 and 2013 and screened water from 345 sites for fecal coliforms and Vibrio cholerae. An international organization/non-governmental organization responsible for construction could be identified for only 56% of water points evaluated. Sixteen percent of water points were non-functional at any given time; 37% had evidence of fecal contamination, with spatial clustering of contaminated sites. Among improved water sources (76% of sites), 24.6% had fecal coliforms versus 80.9% in unimproved sources. Fecal contamination levels increased significantly from 36% to 51% immediately after the passage of Tropical Storm Sandy in October of 2012, with a return to 34% contamination in March of 2013. Long-term sustainability of potable water delivery at a regional scale requires ongoing assessment of water quality, functionality, and development of community-based management schemes...
The 2010 earthquake in Haiti led to thousands of deaths, destruction of drinking and waste-water ... more The 2010 earthquake in Haiti led to thousands of deaths, destruction of drinking and waste-water infrastructure and displacement of millions into temporary camps with little sanitation and hygiene. Even worse, the introduction of Vibrio cholera in October, 2010 resulted in a massive cholera outbreak that spread rapidly throughout Haiti. Once it was recognized that many displaced persons lacked access to water and the outbreak likely resulted from consumption of contaminated surface water, thousands of wells were installed by Non-Government Organizations. However, despite an ongoing epidemic, no water quality data was collected from these wells to verify their safety. To determine if the wells were a source of exposure, 359 sources of drinking water in the Leogane flood basin, located at ground zero of the earthquake, were screened for V. cholerae and fecal coliform bacteria. While no toxigenic strains of V. cholerae were identified, non-toxigenic V. cholerae was isolated from six wa...
Phylodynamic analysis of genome-wide single-nucleotide polymorphism (SNP) data is a powerful tool... more Phylodynamic analysis of genome-wide single-nucleotide polymorphism (SNP) data is a powerful tool to investigate underlying evolutionary processes of bacterial epidemics. The method was applied to investigate a collection of 65 clinical and environmental isolates of Vibrio cholerae from Haiti collected between 2010 and 2012. Characterization of isolates recovered from environmental samples identified a total of four toxigenic V. cholerae O1 isolates, four non-O1/O139 isolates, and a novel nontoxigenic V. cholerae O1 isolate with the classical tcpA gene. Phylogenies of strains were inferred from genome-wide SNPs using coalescent-based demographic models within a Bayesian framework. A close phylogenetic relationship between clinical and environmental toxigenic V. cholerae O1 strains was observed. As cholera spread throughout Haiti between October 2010 and August 2012, the population size initially increased and then fluctuated over time. Selection analysis along internal branches of t...
Extracts in organic solvents (namely methanol, ethanol, ethyl acetate and chloroform) of two medi... more Extracts in organic solvents (namely methanol, ethanol, ethyl acetate and chloroform) of two medicinal plants - Achyranthes aspera and Cassia alata were evaluated for their antibacterial activities against Escherichia coli, Bacillus subtilis, Vibrio cholerae, Salmonella typhi and Staphylococcus aureus. These were carried out by taking the organic extracts of both the leaf and stem parts of the plants at a concentration of 5 mg/ml and their activities were recorded by estimating zones of inhibition as produced by disc-diffusion method on Mueller-Hinton agar media. While neither the leaf nor stem parts of A. aspera in any organic extractions showed antibacterial activity, the methanolic extracts of both the leaf and stem parts of C. alata exhibited antibacterial activity, but only to B. subtilis and S. typhi, and the corresponding MIC values of the leaf extracts were estimated as 1.25 and 1.5 mg/ml respectively. However, the ethanolic extracts of both the stem and leaf parts were foun...
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