Molly M. Freeman

Evolutionary dynamics of Vibrio cholerae O1 following a single-source introduction to Haiti

ABSTRACT Prior to the epidemic that emerged in Haiti in October of 2010, cholera had not been documented in this country. After its introduction, a strain of Vibrio cholerae O1 spread rapidly throughout Haiti, where it caused over 600,000 cases of disease and >7,500 deaths in the first two years of the epidemic. We applied whole-genome sequencing to a temporal series of V. cholerae isolates from Haiti to gain insight into the mode and tempo of evolution in this isolated population of V. cholerae O1. Phylogenetic and Bayesian analyses supported the hypothesis that all isolates in the sample set diverged from a common ancestor within a time frame that is consistent with epidemiological observations. A pangenome analysis showed nearly homogeneous genomic content, with no evidence of gene acquisition among Haiti isolates. Nine nearly closed genomes assembled from continuous-long-read data showed evidence of genome rearrangements and supported the observation of no gene acquisition among isolates. Thus, intrinsic mutational processes can account for virtually all of the observed genetic polymorphism, with no demonstrable contribution from horizontal gene transfer (HGT). Consistent with this, the 12 Haiti isolates tested by laboratory HGT assays were severely impaired for transformation, although unlike previously characterized noncompetent V. cholerae isolates, each expressed hapR and possessed a functional quorum-sensing system. Continued monitoring of V. cholerae in Haiti will illuminate the processes influencing the origin and fate of genome variants, which will facilitate interpretation of genetic variation in future epidemics. IMPORTANCE Vibrio cholerae is the cause of substantial morbidity and mortality worldwide, with over three million cases of disease each year. An understanding of the mode and rate of evolutionary change is critical for proper interpretation of genome sequence data and attribution of outbreak sources. The Haiti epidemic provides an unprecedented opportunity to study an isolated, single-source outbreak of Vibrio cholerae O1 over an established time frame. By using multiple approaches to assay genetic variation, we found no evidence that the Haiti strain has acquired any genes by horizontal gene transfer, an observation that led us to discover that it is also poorly transformable. We have found no evidence that environmental strains have played a role in the evolution of the outbreak strain. Vibrio cholerae is the cause of substantial morbidity and mortality worldwide, with over three million cases of disease each year. An understanding of the mode and rate of evolutionary change is critical for proper interpretation of genome sequence data and attribution of outbreak sources. The Haiti epidemic provides an unprecedented opportunity to study an isolated, single-source outbreak of Vibrio cholerae O1 over an established time frame. By using multiple approaches to assay genetic variation, we found no evidence that the Haiti strain has acquired any genes by horizontal gene transfer, an observation that led us to discover that it is also poorly transformable. We have found no evidence that environmental strains have played a role in the evolution of the outbreak strain.

Characterization of Toxigenic Vibrio cholerae from Haiti, 2010-2011

A virulent clone from Africa or southern Asia was likely introduced at a single time point.

Toxigenic Vibrio cholerae O1 in Water and Seafood, Haiti

During the 2010 cholera outbreak in Haiti, water and seafood samples were collected to detect Vibrio cholerae. The outbreak strain of toxigenic V. cholerae O1 serotype Ogawa was isolated from freshwater and seafood samples. The cholera toxin gene was detected in harbor water samples.

Ebola Virus Diagnostics: The US Centers for Disease Control and Prevention Laboratory in Sierra Leone, August 2014 to March 2015

In August 2014, the Viral Special Pathogens Branch of the US Centers for Disease Control and Prevention established a field laboratory in Sierra Leone in response to the ongoing Ebola virus outbreak. Through March 2015, this laboratory tested >12 000 specimens from throughout Sierra Leone. We describe the organization and procedures of the laboratory located in Bo, Sierra Leone.

Cholera in the United States, 2001–2011: a reflection of patterns of global epidemiology and travel

US cholera surveillance offers insight into global and domestic trends. Between 2001 and 2011, 111 cases were reported to the Centers for Disease Control and Prevention. Cholera was associated with international travel in 90 (81%) patients and was domestically acquired in 20 (18%) patients; for one patient, information was not available. From January 2001 to October 2010, the 42 (47%) travel-associated cases were associated with travel to Asia. In October 2010, a cholera epidemic started in Haiti, soon spreading to the Dominican Republic (Hispaniola). From then to December 2011, 40 (83%) of the 48 travel-associated cases were associated with travel to Hispaniola. Of 20 patients who acquired cholera domestically, 17 (85%) reported seafood consumption; 10 (59%) ate seafood from the US Gulf Coast. In summary, an increase in travel-associated US cholera cases was associated with epidemic cholera in Hispaniola in 2010-2011. Travel to Asia and consumption of Gulf Coast seafood remained important sources of US cholera cases.

Household Water Treatment Uptake during a Public Health Response to a Large Typhoid Fever Outbreak in Harare, Zimbabwe

Locally manufactured sodium hypochlorite (chlorine) solution has been sold in Zimbabwe since 2010. During October 1, 2011-April 30, 2012, 4,181 suspected and 52 confirmed cases of typhoid fever were identified in Harare. In response to this outbreak, chlorine tablets were distributed. To evaluate household water treatment uptake, we conducted a survey and water quality testing in 458 randomly selected households in two suburbs most affected by the outbreak. Although 75% of households were aware of chlorine solution and 85% received chlorine tablets, only 18% had reportedly treated stored water and had the recommended protective level of free chlorine residuals. Water treatment was more common among households that reported water treatment before the outbreak, and those that received free tablets during the outbreak (P < 0.01), but was not associated with chlorine solution awareness or use before the outbreak (P > 0.05). Outbreak response did not build on pre-existing prevention programs.

Shifts in Geographic Distribution and Antimicrobial Resistance during a Prolonged Typhoid Fever Outbreak — Bundibugyo and Kasese Districts, Uganda, 2009–2011

Background Salmonella enterica serovar Typhi is transmitted by fecally contaminated food and water and causes approximately 22 million typhoid fever infections worldwide each year. Most cases occur in developing countries, where approximately 4% of patients develop intestinal perforation (IP). In Kasese District, Uganda, a typhoid fever outbreak notable for a high IP rate began in 2008. We report that this outbreak continued through 2011, when it spread to the neighboring district of Bundibugyo. Methodology/Principal Findings A suspected typhoid fever case was defined as IP or symptoms of fever, abdominal pain, and ≥1 of the following: gastrointestinal disruptions, body weakness, joint pain, headache, clinically suspected IP, or non-responsiveness to antimalarial medications. Cases were identified retrospectively via medical record reviews and prospectively through laboratory-enhanced case finding. Among Kasese residents, 709 cases were identified from August 1, 2009–December 31, 2011; of these, 149 were identified during the prospective period beginning November 1, 2011. Among Bundibugyo residents, 333 cases were identified from January 1–December 31, 2011, including 128 cases identified during the prospective period beginning October 28, 2011. IP was reported for 507 (82%) and 59 (20%) of Kasese and Bundibugyo cases, respectively. Blood and stool cultures performed for 154 patients during the prospective period yielded isolates from 24 (16%) patients. Three pulsed-field gel electrophoresis pattern combinations, including one observed in a Kasese isolate in 2009, were shared among Kasese and Bundibugyo isolates. Antimicrobial susceptibility was assessed for 18 isolates; among these 15 (83%) were multidrug-resistant (MDR), compared to 5% of 2009 isolates. Conclusions/Significance Molecular and epidemiological evidence suggest that during a prolonged outbreak, typhoid spread from Kasese to Bundibugyo. MDR strains became prevalent. Lasting interventions, such as typhoid vaccination and improvements in drinking water infrastructure, should be considered to minimize the risk of prolonged outbreaks in the future.

Vibriosis, not cholera: toxigenic Vibrio cholerae non-O1, non-O139 infections in the United States, 1984-2014.

Toxigenic strains of Vibrio cholerae serogroups O1 and O139 have caused cholera epidemics, but other serogroups - such as O75 or O141 - can also produce cholera toxin and cause severe watery diarrhoea similar to cholera. We describe 31 years of surveillance for toxigenic non-O1, non-O139 infections in the United States and map these infections to the state where the exposure probably originated. While serogroups O75 and O141 are closely related pathogens, they differ in how and where they infect people. Oysters were the main vehicle for O75 infection. The vehicles for O141 infection include oysters, clams, and freshwater in lakes and rivers. The patients infected with serogroup O75 who had food traceback information available ate raw oysters from Florida. Patients infected with O141 ate oysters from Florida and clams from New Jersey, and those who only reported being exposed to freshwater were exposed in Arizona, Michigan, Missouri, and Texas. Improving the safety of oysters, specifically, should help prevent future illnesses from these toxigenic strains and similar pathogenic Vibrio species. Post-harvest processing of raw oysters, such as individual quick freezing, heat-cool pasteurization, and high hydrostatic pressurization, should be considered.

Canonical Single Nucleotide Polymorphisms (SNPs) for High-Resolution Subtyping of Shiga-Toxin Producing Escherichia coli (STEC) O157:H7.

The objective of this study was to develop a canonical, parsimoniously-informative SNP panel for subtyping Shiga-toxin producing Escherichia coli (STEC) O157:H7 that would be consistent with epidemiological, PFGE, and MLVA clustering of human specimens. Our group had previously identified 906 putative discriminatory SNPs, which were pared down to 391 SNPs based on their prevalence in a test set. The 391 SNPs were screened using a high-throughput form of TaqMan PCR against a set of clinical isolates that represent the most diverse collection of O157:H7 isolates from outbreaks and sporadic cases examined to date. Another 30 SNPs identified by others were also screened using the same method. Two additional targets were tested using standard TaqMan PCR endpoint analysis. These 423 SNPs were reduced to a 32 SNP panel with the almost the same discriminatory value. While the panel partitioned our diverse set of isolates in a manner that was consistent with epidemiological data and PFGE and MLVA phylogenies, it resulted in fewer subtypes than either existing method and insufficient epidemiological resolution in 10 of 47 clusters. Therefore, another round of SNP discovery was undertaken using comparative genomic resequencing of pooled DNA from the 10 clusters with insufficient resolution. This process identified 4,040 potential SNPs and suggested one of the ten clusters was incorrectly grouped. After its removal, there were 2,878 SNPs, of which only 63 were previously identified and 438 occurred across multiple clusters. Among highly clonal bacteria like STEC O157:H7, linkage disequilibrium greatly limits the number of parsimoniously informative SNPs. Therefore, it is perhaps unsurprising that our panel accounted for the potential discriminatory value of numerous other SNPs reported in the literature. We concluded published O157:H7 SNPs are insufficient for effective epidemiological subtyping. However, the 438 multi-cluster SNPs we identified may provide the additional information required.

Typhoid fever acquired in the United States, 1999–2010: epidemiology, microbiology, and use of a space–time scan statistic for outbreak detection

Although rare, typhoid fever cases acquired in the United States continue to be reported. Detection and investigation of outbreaks in these domestically acquired cases offer opportunities to identify chronic carriers. We searched surveillance and laboratory databases for domestically acquired typhoid fever cases, used a space-time scan statistic to identify clusters, and classified clusters as outbreaks or non-outbreaks. From 1999 to 2010, domestically acquired cases accounted for 18% of 3373 reported typhoid fever cases; their isolates were less often multidrug-resistant (2% vs. 15%) compared to isolates from travel-associated cases. We identified 28 outbreaks and two possible outbreaks within 45 space-time clusters of ⩾2 domestically acquired cases, including three outbreaks involving ⩾2 molecular subtypes. The approach detected seven of the ten outbreaks published in the literature or reported to CDC. Although this approach did not definitively identify any previously unrecognized outbreaks, it showed the potential to detect outbreaks of typhoid fever that may escape detection by routine analysis of surveillance data. Sixteen outbreaks had been linked to a carrier. Every case of typhoid fever acquired in a non-endemic country warrants thorough investigation. Space-time scan statistics, together with shoe-leather epidemiology and molecular subtyping, may improve outbreak detection.