Cholera is a disease endemic in many developing countries, and the causative agent, Vibrio cholerae, is autochthonous to estuaries and river systems worldwide (1). The recent outbreak of cholera in Haiti is a stark reminder of how rapidly a cholera epidemic can emerge and spread through a population. On January 12, 2010, a devastating 7.0 Mw earthquake struck Haiti, and, in the ensuing 12 d, ∼52 aftershocks with a magnitude of 4.5 Mw or greater were recorded. It is estimated that more than 250,000 people died, ∼300,000 were injured, and 1 million were rendered homeless. A very hot summer season followed. Amid the clearing of rubble and rebuilding of infrastructure, the first case of cholera was diagnosed on October 21, 2010, and then the disease quickly erupted throughout Haiti. On November 5, 2010, the full force of Hurricane Tomas skirted the island, causing torrential rains and severe flooding. Cholera, not officially reported in Haiti since 1960 (2), erupted in epidemic proportions (3), and, in just 1 y after the initial cases, cholera has claimed more than 6,500 lives and sickened approximately 500,000 people (4). The series of events of 2010 clearly created a “perfect storm” for cholera, with assignment of attribution remaining controversial (4).
Identical pulsed field gel electrophoresis patterns for 13 bacterial isolates recovered from Haitian patients with cholera led to a conclusion that the Haitian isolates derived from a strain similar to V. cholerae isolated in South Asia (3). From a limited comparison of genomes, Chin et al. concluded the Haitian cholera epidemic was most likely a result of an introduction of a strain from a distant geographic location and derived from human activity (5). A United Nations Independent Panel of Experts, in a report on the 2010 cholera outbreak in Haiti, concluded that the Haiti cholera outbreak was a result of contamination of water upstream in the Artibonite River with a pathogenic strain of a South Asian type of V. cholerae (6). Another report (7) that used whole-genome sequence typing of Nepalese isolates and compared them with a few previously sequenced V. cholerae strains, interpreted the results as indicating that Nepal was the likely origin of the Haitian outbreak. A rebuttal, however, was published, which pointed out that South Asia, not Nepal, might be the origin of the Haitian cholera outbreak strains, stating correctly that proper attribution cannot be accomplished by an exclusionary approach (8). Notably, none of the Nepalese soldiers had been recorded to be ill during the outbreak in Haiti (4); therefore, involvement of Nepalese peacekeepers remains controversial (9). A further study that used whole-genome phylogeny and core genome SNPs (8) showed the Haiti outbreak strain to be genetically related to strains originating in India and Cameroon. Like earlier studies, however, the authors concluded that a definitive genetic origin for the outbreak in Haiti must remain speculative (8).
Although the molecular and genomic data in the previous reports cited here have been interpreted as being consistent with V. cholerae being introduced to Haiti by outside visitors (5, 7, 8), a definitive statement of source attribution cannot yet be made. To assess source attribution, a qualified reference database (including recent strains from Nepal, India, and Cameroon, and related V. cholerae strains from concurrent epidemics) representative of global phylogenetic diversity as well as well-documented population diversity within the heterogeneous clade and its near neighbor comparators, is critical. In the present study, the genomic diversity of Haitian isolates was examined, and these data are presented to provide a broader perspective of the complexity of the species, V. cholerae, particularly of those isolates from Haiti, in an effort to contribute to the understanding of the origin of the outbreak as indigenous or introduced.
Results and Discussion
Clinical samples from 81 patients who showed symptoms of cholera with varying severity were collected from 18 towns across eight Arrondissements of Haiti (Fig. 1 and Table S1), spanning 3 wk in November 2010, very early in the outbreak. Bacteriological analysis identified V. cholerae O1 and V. cholerae non-O1/O139 as pathogen in 48% (n = 39) and 21% (n = 17) of the samples, respectively. V. cholerae O1 and non-O1/O139 were cocultured from 7% of the O1 positive samples. Isolation of V. cholerae non-O1/O139, in addition to toxigenic V. cholerae O1, indicated that at least two disparate V. cholerae populations coexisted early in the outbreak. From the remaining 31% (n = 25) of the clinical samples, other enteric pathogens were also cultured, and subsequent analysis of a portion of the latter indicated the presence of opportunistic pathogens including Aeromonas caviae, Aeromonas hydrophila, and Proteus mirabilis. Environmental samples were also collected from two different towns in Haiti concurrent with the epidemics; bacteriological analysis of these samples identified V. cholerae non-O1/O139 from each of those samples.
Source and distribution of isolates collected during the 2010 Haitian cholera outbreak. Yellow and blue circles indicate sites where clinical and environmental strains were collected, respectively. Left: Distribution of strains with respect to different locations of sample collection in Haiti.
Multilocus Variable-Number Tandem Repeat Analysis.
Multilocus variable-number tandem repeat (VNTR) analysis (MLVA), a useful tool for tracking V. cholerae (10) and resolving distinct populations of clinical isolates from different geographic regions (11, 12), was used to examine the clinical and environmental V. cholerae O1 isolates from Haiti and other geographical locations (Table 1). Altered El Tor V. cholerae circulating in Southeast Asia has been reported to be a stable VNTR genotype over the years [Bangladesh, 2001–2005 (12), and India, 2004–2007 (11)], and our typing of the 9.3.6.n.n VNTR genotype supported the stability of that genotype. Haitian V. cholerae O1 (HCO1) strains, on the contrary, displayed a nearly identical VNTR genotype (Table 1), forming a tight clonal complex, as also reported previously (13). Investigation of V. cholerae strains from Zimbabwe, Zambia, Mexico, and Thailand confirmed, the Haitian V. cholerae strains contained a genetically distinct genotype (8.3.6.n.n) not observed previously, representing a unique VNTR genotype (Table 1). The MLVA data provide two valuable pieces of information. First, clonal transmission of VNTR alleles, or the limited VNTR diversity among the Haitian outbreak strains, indicates either a possible single source of contamination, in agreement with other findings (13); or a single clone of V. cholerae responsible for the epidemic. Secondly, Haitian strains exhibit a genetically distinct MLVA profile. Environmental V. cholerae non-O1/O139 isolates exhibited heterogeneous VNTR genotypes, with variability at all five loci (Table 1). Notable, however, was the observation that the clinical V. cholerae non-O1/O139 isolates revealed an identical VNTR genotype, akin to, but distinct from, the clinical V. cholerae O1 isolates.
VNTR genotypes of a subset of V. cholerae strains isolated from Haiti and other geographic locations
Genomic Relatedness of Haitian Strains with Reference V. cholerae Strains.
Whole-genome sequencing of 47 HCO1, 29 V. cholerae non-O1/O139, and eight reference strains of V. cholerae isolated outside Haiti in concurrent epidemics including in Bangladesh (CP1048, 2010) and Thailand (CP1042, 2010), as well as older epidemics (Table S1), was performed for this study. Genome comparisons of these 76 genomes and an additional 27 V. cholerae genomes available in the public database were conducted to ascertain the degree of genomic diversity within V. cholerae isolates from the same epidemics.
The Haitian genome sequences were found to be genetically similar to the recently emerged V. cholerae Altered El Tor strains CP1038 (Zimbabwe, 2009), CP1048 and CP1050 (Bangladesh, 2010), CP1041 (Zambia, 2004), and CP1042 (Thailand, 2010). These genomes belong to the seventh-pandemic clade, possessing a genomic backbone representative of V. cholerae O1 El Tor and V. cholerae O139, i.e., phylocore genome 1 clade (14, 15) with variations in their mobile genetic elements (MGEs) (Table S2). MGEs encode toxins, antibiotic resistance, virulence factors, and a wide range of proteins with as yet-unknown function. MGEs are resident in the natural genetic reservoir of V. cholerae and related Vibrio spp., including Vibrio parahaemolyticus and Vibrio mimicus (14, 15). Thus, within a dynamic microbial gene pool, in which horizontal gene transfer is a significant driver of microbial evolution (16), MGEs play a pivotal role in the genesis of diversity and hypervariability of Vibrio genomes (14, 15). The MGE catalogue of the HCO1 strains demonstrates that they share genetically similar genomic and pathogenic islands (n = 18) with the recently isolated Altered El Tor V. cholerae strains from South Asia and Africa (Table S2). Like the concurrent epidemic strains, CTXΦ genes were located in the large chromosome (C-I) of the HCO1 isolates (Table S2). RS1 was detected in most of the HCO1 strains, but four strains appeared to lack RS1 (Table S2), as does strain (CP1041) from a 2004 epidemic in Zambia. In addition, all HCO1 isolates, except HC49A2, which also lacked RS1, harbor TLC (Toxin-linked cryptic plasmid, a genetic element that plays some role in the biology of CTXΦ, perhaps facilitating its acquisition or its replication). As anticipated, none of the Haitian V. cholerae non-O1/O139 strains had CTXΦ, RS1, or TLC.
Two genomic islands (GIs) in two HCO1 strains (GI-16 in AELH.1792 and GI-120 in HC-77A1) were located in the same insertion site (VCA0569-VCA0570) in the small chromosome (C-II) but absent in all concurrent epidemic strains examined in this study. GI-16 is a 6.6-kb island engendering Vibrio VSK prophage, a pre-CTX prophage, integration of which has previously been described in V. cholerae O139 MO10 (in C-I) (15) and non-O1/O139 AM-19226 and 1587 (in C-II) (16), but heretofore not reported in V. cholerae O1 to our knowledge. Further inspection identified GI-16, with a genetically similar set of coding sequences (CDS) on C-I (VC1451-VC1456), in six of the Haitian non-O1/O139 strains, including one strain (HC-2A1) containing GI-16 at both the C-I and C-II insertion sites (Table S2). GI-120, inserted at the same insertion site in HC-77A1, is a 31.5-kb island with 30 CDSs, encoding different transporters and stress response protein. Our search for this GI identified another Haitian strain, HC-78A1, a clinical non-O1/O139 isolate, harboring this GI at the same chromosomal location. Subsequent analysis indicated GI-120 might be the result of rearrangement of the C-I loci VC1416 to VC1446 into C-II. In addition, all HCO1 strains contained the 10 GIs (GI-01 to GI-10) previously reported in V. cholerae O1 (Table S2). In contrast, the 29 clinical and environmental V. cholerae non-O1/O139 strains revealed a number of GIs (n = 40) encoding genes for virulence, survival, stress response, and metabolism, and distributed across both chromosomes. The environmental strains possessed a substantially larger repertoire of GIs (Table S2).
Variations in CTXΦ-RS1.
CTXΦ present in toxigenic V. cholerae consists of two functionally distinct gene clusters, core and RS2 region (17, 18). The core contains ctxAB, encoding cholera toxin (CT), together with five other genes—psh, cep, pIIICTX, ace, and zot—required for phage morphogenesis. RS2 encodes proteins with roles in replication (RstA), integration (RstB), and regulation (RstR) of site-specific recombination of CTXΦ (17, 19). In toxigenic V. cholerae El Tor strains, a satellite phage, RS1, invariably is present and located adjacent to CTXΦ. It is similar to RS2 except that it contains an additional gene, rstC, that encodes an antirepressor protein, promoting transcription of CTXΦ genes required for generating infectious particles (19). The CTXΦ and RS1 element array in the Haitian cholera isolates are similar, but not identical, to those found in the Altered El Tor V. cholerae strains, i.e., CIRS101.
We observed a single amino acid deletion (delta S), a GTA deletion at nucleotides 77 to 79 in rstB of the HCO1 strains (Table 2). This deletion, a unique feature for a classical biotype of V. cholerae (20), was not detected in any of the Altered El Tor, Hybrid, or El Tor variants of V. cholerae, except V. cholerae CP1048 (Bangladesh, 2010) and CP1032 (Mexico, 1991). Interestingly, V. cholerae INDRE 91/1 (Mexico, 1991) and RC27 (Indonesia, 1991) contained the GTA deletion, underscoring this deletion is not novel to Western Hemisphere isolates. Moreover, PCR amplification and sequence analysis of rstB gene identified unique polymorphisms (A to G at nucleotide 192 and G to A at nucleotide 108). The former was found only in the HCO1 strains, whereas the latter was also detected in the Mexico strains, V. cholerae INDRE91/1 and CP1032, suggesting the G-to-A polymorphism may be specific to Western Hemisphere strains, a conclusion that requires additional data.
Variation was also detected in the number of ToxR binding repeats (TTTTGAT) between zot and ctxA. V. cholerae. ToxR is a global transcriptional regulator of virulence gene expression, and this repeated sequence is required for ToxR binding and activation of the ctxAB promoter. The ToxR-binding site is located immediately upstream of ctxAB, and affinity of ToxR binding is influenced by the repeat sequences (21). The HCO1 strains contained five copies of this repeat (Table 2), compared with three or four copies in other Altered El Tor, Hybrid, or El Tor variants of V. cholerae, including recent epidemic strains (CP1048, CP1038, and CP1032), and seven copies were found in the classical biotype of V. cholerae O395. The presence of an increased number of ToxR binding repeats has been hypothesized to correlate with a severe form of cholera (22, 23), which may be the case in the Haitian epidemic (24).
A further feature of the Haitian strains was the presence of three mutations in ctxB compared with two in Altered El Tor Hybrid or El Tor variant strains of V. cholerae O1 (24). The same ctxB polymorphisms in V. cholerae have previously been reported for strains from Kolkata and Orissa, India, and most recently in V. cholerae isolates from Nigeria, Cameroon, Kenya, Sudan, Ethiopia, and Nepal (25, 26). Taken together, these data suggest that the ancestor to the strains responsible for the Haitian outbreak successfully used genetic changes in CTXΦ and RS1 element to affect disease severity, an attribute of the classical biotype, as well as better environmental fitness, an attribute of the El Tor biotype (5, 9⇓–11).
Sites of nucleotide variation in CTX prophages and RS1 elements
Vibrio Pathogenicity Island-1.
Vibrio pathogenicity island-1 (VPI-1) codes for the toxin coregulated pilus involved in biofilm formation, attachment to chitinous organisms, and as the receptor for CTXφ (27). This island was found in all HCO1 isolates but not in clinical or environmental V. cholerae non-O1/O139 isolates. Interestingly, although devoid of VPI-1, three different GIs were identified at the VPI-1 site in 85% of non-O1/O139 isolates (Table S3). GI-41 was identified at the VPI-1 locus in one environmental and 15 clinical isolates, whereas other GIs, GI-102 and GI-81, were identified in other environmental isolates (Table S3). It is of interest that GI-81 represents a part of the Vibrio Seventh Pandemic (VSP) Island II (VSP-II) but is inserted at the VPI-1 site (Tables S2 and S3).
Vibrio Pathogenicity Island-2.
Most of the genomes have encoded sequences with at least some homology to the Vibrio pathogenicity island-2 (VPI-2) inserted at the canonical locus for this island (VC1758 and VC1810) (15). Whereas all V. cholerae O1 strains contained a complete VPI-2, many non-O1/O139 strains, both clinical and environmental, contained only the sialidase sequence of the sialic acid metabolism region of VPI-2. That is, the VPI-2 locus in non-O1/O139 strains lack most of the VPI-2 ORFs, including the type 1 restriction modification system and mu-like phage regions (Table S3). Interestingly, the sialidase has been shown to unmask the GM1 gangliosides of human intestinal epithelial cells, thus making them more available to CT (28). However, because many of these strains do not encode CT, the high level of conservation of sialidase among these strains suggests an additional role for sialidase in the life cycle of Vibrio. Notably, the clinical non-O1/O139 isolate HC1A2 contained GI-121 inserted at VPI-2 site and part of GI-09 encoding genes responsible for response regulator, autolysin sensor kinase, and ABC-type transport system. An environmental isolate, V. cholerae HE25, encoded a type III secretion system within the VPI-2 region. The type III secretion systems most frequently found in V. cholerae serogroups non-O1/non-O139 induce severe diarrhea in cholera infection models (29), and this secretion system has been reported in V. cholerae strains that encode and express the O1-antigen (15). Our results are consistent with previous studies that showed VPI-2 to be highly heterogeneous in clinical and environmental strains (15, 28).
All HCO1, but none of the Haitian V. cholerae non-O1/O139 genomes examined in this study, carried VSP-I. Interestingly, an environmental V. cholerae non-O1/O139, HE16, carried a previously uncharacterized GI at the VSP-I insertion loci. Similarly, all HCO1 carried a variant VSP-II, first reported in V. cholerae O1 CIRS101, although none of the V. cholerae non-O1/O139 were found to carry this or any other variant at the VSP-II locus (Table S3). The VSP-II island in O1 strains was inserted at the usual insertion locus in the genome and, as observed in V. cholerae CIRS101, revealed many deletions compared with the VSP-II in other V. cholerae O1 seventh pandemic strains described previously (30).
Integrated Conjugated Elements.
Integrated conjugated elements (ICEs) found in HCO1 strains were compared with ICEVchBan5 and ICEVchMoz in V. cholerae O1 CIRS101 and B33, respectively. Analysis of their genetic organization revealed HCO1 ICE is >99% similar to ICEVchBan5, differing only by 5SNPS (31). In contrast, the majority of the Haitian V. cholerae non-O1/O139 strains carry an ICE sequence variant; only three strains lacked an insertion at this site (Table S3). All the ICE insertions encode an integrase gene of the P4 integrase family and integrate at prfC on C-I. Minor differences between these elements and ICEVchBan5 were observed at the junctions between sequence contigs. In contrast, the ICE found in Haitian clinical V. cholerae non-O1/O139 was distinct from any ICE reported to date, notably lacking the cluster of CDSs encoding antibiotic resistance. Although this might be expected in view of the higher variability observed in ICE present in V. cholerae non-O1/O139 from other geographical areas (32), it is interesting that the same element is present in each of the Haitian V. cholerae non-O1/O139 strains. Even more intriguing is the fact that these V. cholerae non-O1/O139 strains were of clinical origin and in 21% the only isolate cultured from the patient.
Mutations in gyrA and parC.
The Haitian V. cholerae O1 isolates were resistant to sulfisoxazole, trimethoprim-sulfamethoxazole, furazolidone, streptomycin, and nalidixic acid, and displayed reduced susceptibility to ciprofloxacin (3, 5). The strains were sensitive to azithromycin and tetracycline. Analysis of the gyrA and parC (VC1258 and VC2430, respectively, in Table S3) genes revealed two point mutations in the Haitian strains, i.e., a ser83ile substitution in gyrA and a ser85leu substitution in parC. Both point mutations are associated with quinolone resistance in clinical V. cholerae and have been reported in India and most recently in Nigeria and Cameroon (33⇓⇓⇓–37). We observed the same point mutations in gyrA and parC in recent isolates from Zimbabwe (CP1038), Thailand (CP1042), and Bangladesh (CP1048). In contrast, whereas V. cholerae O1 CIRS101 (Bangladesh, 2002) and CP1041 (Zambia, 2004) contained the same point mutation at gyrA, neither strain harbored the parC mutation, offering evidence of similarity with, but not identity to, V. cholerae in Asia and Africa.
The phylogeny of the Haitian V. cholerae isolates was inferred by constructing a genome-relatedness neighbor-joining tree by using homologous alignment of 992 orthologous protein-coding genes (∼989,552 bp) of 96 V. cholerae genomes, as a strict measure of the core V. cholerae genome. The evolutionary tree (Fig. 2A) corroborated the previously described 1.7-million-bp phylogeny of V. cholerae (15), showing serogroups of V. cholerae evolving into distinct evolutionary lineages. Significantly, 26 of 29 V. cholerae non-O1/O139 strains included in our phylogenomic analysis clustered with toxigenic V. cholerae O1 strains (i.e., Amazonia and TM11079-80; Fig. 2A) implicated in sporadic cases and severe epidemics originating in the Western Hemisphere. The phylogeny is concordant with the observation that non-O1/O139 strains isolated from the Haitian clinical samples harbor a battery of virulence/fitness factors organized on MGEs. That is, despite being non-O1/O139, these strains harbor the genomic backbone similar to some toxigenic V. cholerae O1, explaining the coexistence of non-O1/O139 with V. cholerae O1. Furthermore, the clinical non-O1/O139 isolates formed two closely related clusters (HC-1 and HC-2), with the majority of the non-O1/O139 isolates clustering together in HC-2 (Fig. 2B). Both clinical and environmental isolates clustered together, along with 1991 and 1980 V. cholerae O1 isolates from Brazil, indicating potential indigenous origin of these strains.
Neighbor-joining trees showing phylogenetic relationships based on 992 orthologous protein-coding genes (∼989,552 bp) of (A) 96 V. cholerae genomes representing diverse serogroups (strains with blue and green color represent Haitian environmental and clinical isolates, respectively), (B) V. cholerae non-O1/O139 strains representing clinical non-O1/O139 group 2, and (C) V. cholerae O1 and O139 strains representing the seventh pandemic cluster.
The HCO1 strains, on the contrary, clustered with other seventh pandemic V. cholerae strains in a single monophyletic clade (Fig. 2A). A tree constructed with only seventh pandemic strains (Fig. 2C) showed clustering of the Haitian strains together, as well as with clinical isolates from Zimbabwe (CP1038) and Bangladesh (CP1048 and CP1050), an observation in agreement with the recent report of shared ancestry of Haiti, India, and Cameroon isolates (8). HCO1 isolates subdivided into several closely related clusters within a tight monophyletic clade indicating stochastic biological transmission events resulting in genetic drift among the strains isolated during the first 3 wk of the outbreak. Genetic drift would account for radiation of ∼50% of the isolates away from the basal Haitian isolates. To better demonstrate the heterogeneity of these clonal subpopulations, principal component analysis (PCA), a multivariate method useful for exploratory cluster visualization, was used. Based on the divergence of average nucleotide and tetra nucleotide frequency matrix using R, PCA analysis showed the Haitian strains formed a heterogeneous cloud of clonal subpopulations (Fig. S1), indicating that two closely related isolates from Bangladesh (CP1048) and Zimbabwe (CP1048) hard to exclude as maximum variations observed among the various Haitian strains must be included (Fig. S1).
Relatedness of Outbreak and Reference Isolates by Whole-Genome Mapping.
Whole-genome maps were created for a set of Haitian V. cholerae isolates and isolates from concurrent epidemics elsewhere. Comparison of the whole-genome maps showed all HCO1 outbreak strains to be identical within the limits of the mapping technology resolution, suggesting the outbreak to be clonal. Detailed whole-genome map comparisons of outbreak isolates with closely related reference isolates CP1048 and CP1038 revealed substantial similarity, yet differences were observed (Fig. 3 and Fig. S2). Comparison of these regions with annotated sequences of a previously studied isolate, AE-1786, showed that, although most of the HCO1 and CP1048 (Bangladesh, 2010) contained TLC of ∼18 kb, two strains AE-1786 (Haiti, 2010) and CP1038 (Zimbabwe, 2009), displayed a TLC of ∼13 kb (Fig. 3A). Distinct polymorphism was also observed in the mannose-sensitive hemagglutinin (MSHA; Fig. 3B), a surface pilus contributing the ability of V. cholerae to adhere to plankton (38). Detailed whole-genome map comparisons demonstrated three Haitian strains—AE-1786, HC61A1, and HC46A1—harbored an ∼18-kb tRNA cluster in MSHA, whereas other HCO1 and two closely related reference isolates—CP1048 and CP103—contained an approximately 24-kb cluster (Fig. 3B). A larger insert (∼27 kb) was identified at the ICE locus in Haitian strain HC20A2, compared with the ∼18-kb insert found in other HCO1 strains (Fig. 3D), as well as CP1048 and CP1038.
Polymorphisms in Haitian outbreak and reference isolates as determined by whole-genome mapping. Polymorphism detected in (A) TLC element, (B) MSHA gene clusters, (C) tRNA region, and (D) ICE element. Highlighted by the blue colored crossed line box are regions where polymorphisms were detected.
Genome similarity clustering (Fig. 4) of whole-genome maps for both chromosomes showed the outbreak isolates did not differ substantially from CP1038 and CP1048. However, other strains from Thailand (CP1042), Mexico (CP1032 and 1033), and Zambia (CP1041) differed by >2% and therefore clustered separately. All clinical and environmental non-O1/O139 strains clustered in a monophyletic group. The genome similarity clustering confirmed heterogeneity of the V. cholerae non-O1/O139, as reported previously (38).
Citation: Orata FD, Keim PS, Boucher Y (2014) The 2010 Cholera Outbreak in Haiti: How Science Solved a Controversy. PLoS Pathog 10(4): e1003967. https://doi.org/10.1371/journal.ppat.1003967
Editor: Joseph Heitman, Duke University Medical Center, United States of America
Published: April 3, 2014
Copyright: © 2014 Orata et al. 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.
Funding: This work was supported by the Integrated Microbial Biodiversity Program of the Canadian Institute for Advanced Research (CIFAR) and the Natural Sciences and Engineering Research Council of Canada (NSERC) to FDO and YB. PSK was supported by the Northern Arizona University Cowden Endowment for Microbiology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The 2010 Earthquake and Cholera Outbreak in Haiti
On January 12, 2010, a catastrophic 7.0 magnitude earthquake struck Haiti, affecting 3,500,000 people , . This severely damaged an already marginal public sanitation system, creating ideal conditions for outbreaks of major infectious diseases. In October 2010, nine months after the earthquake, an outbreak of cholera started, which quickly spread all across the country . As of January 7, 2014, 8,534 deaths and 697,256 cholera cases have been reported by the Haitian Ministry of Public Health and Population . Prior to 2010, there was no reported history of cholera in Haiti, despite devastating outbreaks in the Caribbean region in the 19th century . Many wondered where the cholera in Haiti came from. Two hypotheses as to its origin were presented. The climatic hypothesis argued that nonpathogenic Vibrio cholerae, indigenous in the coastal waters of Haiti, was given the right environmental circumstances and evolved into a pathogenic strain . On the other hand, the human transmission hypothesis suggested that cholera was introduced to Haiti by individuals infected in a foreign country.
Cholera and V. cholerae
Cholera, caused by V. cholerae, is a disease characterized by very severe diarrhea and dehydration, which can lead to death in less than 48 hours if left untreated. Cholera is treatable through oral rehydration salt solutions, intravenous fluids, or antibiotics, depending on severity . Ingestion of contaminated water is the main vehicle for human infection. The principal virulence determinant is the potent cholera toxin, encoded by the ctxAB genes on the bacteriophage CTXφ  found in toxigenic V. cholerae genomes. The toxin, together with other virulence factors, leads to the harmful effects of the V. cholerae infection (Figure 1). These auxiliary virulence factors are encoded in clusters of genes called genomic islands, which are acquired by environmental V. cholerae through horizontal gene transfer  (Figure 1). It is also important to note that infection can be asymptomatic, and these cases play a major role in the transmission of the disease . V. cholerae is of major public health concern because of its potential to cause pandemics. Seven such pandemics have been recorded since 1817, when cholera first spread beyond the Indian subcontinent, all presumably caused by V. cholerae belonging to the O1 serogroup. V. cholerae of the classical biotype dominated the previous six pandemics and was replaced by the El Tor biotype in the currently ongoing seventh pandemic, which originated in Southeast Asia in 1961 . In 1992, a new serogroup of V. cholerae, O139, was first identified after causing cholera epidemics in India and Bangladesh . Cholera has been eliminated from industrialized countries by efficient water and sewage treatments but not in less-developed countries with poor water sanitation.
Figure 1. Steps in the evolution of the seventh pandemic Vibrio cholerae.
Environmental V. cholerae indigenous in coastal waters can harbor genomic islands (GIs) by horizontal gene transfer, rendering it pathogenic. Pathogenesis of toxigenic (toxin-producing) V. cholerae critically depends on the production of the cholera toxin, which is responsible for the cholera symptoms, and the toxin-coregulated pilus (TCP). The genes for the cholera toxin (ctx) are from the filamentous bacteriophage, CTXφ, that has been incorporated into the genome. The genes in the TCP island encode factors necessary for the colonization of the small intestine in the human host after ingestion of contaminated water. Additionally, seventh pandemic strains are distinguishable from pre-seventh pandemic strains due to the acquisition of additional GIs, the Vibrio seventh pandemic (VSP) islands.
Initial Studies Support the Human Transmission Hypothesis
Rumors spread on October 27, 2010, pointing blame for the outbreak at the United Nations Stabilization Mission in Haiti (MINUSTAH) troops from Nepal who had recently set up camp in Meille, a small village 2 km south of Mirebalais (Figure 2A). This followed revelations by news reporters showing improper sewage waste disposal in the camp , .
Figure 2. How the Haiti cholera outbreak started.
(A) MINUSTAH troops from Nepal were stationed in Haiti starting on October 8, 2010, and set up camp in Meille (red circle). Improper disposal of sewage led to the contamination of the Meille tributary, which connects downstream to the Latem River (red arrow). The first case of cholera occurred on October 12 along the Latem River in Mirebalais (orange circle), 2 km north of Meille. Water from the Latem River enters the Artibonite River (orange arrow), the major river that spans across Haiti, which flows downstream to St. Marc (blue arrow). The Artibonite River played a significant role in the rapid spread of cholera. During the early onset of the epidemic, reported cases were linked to proximity with the river. (B) A chronological timeline of events involving the Haiti cholera outbreak from July to December 2010.
The stool samples collected by the Haiti National Public Health Laboratory from cholera patients at the start of the outbreak were sent to the Centers for Disease Control and Prevention (CDC) for analysis. On November 13, the CDC reported that V. cholerae El Tor O1 was isolated from the samples and independent isolates were indistinguishable by multiple rapid phenotypic and molecular characterization methods, suggesting that a single strain caused the outbreak and was likely introduced into Haiti in one event .
A study by Piarroux and colleagues made use of all available epidemiological data, checking hospital records, doing field surveys, and applying statistics for spatiotemporal analysis, to trace the source and spread of the outbreak . The findings of their investigation confirmed the news reporters' claims. Based on all gathered evidence, they put together a likely scenario – the MINUSTAH camp contaminated the Meille tributary with fecal matter by their unsanitary practice of sewage drainage, and the Meille tributary connects downstream to the Latem River that goes through the town of Mirebalais, the site of the first reported cholera case . The Latem River eventually connects to the Artibonite River, the longest as well as the most important river that spans Haiti (Figure 2A). The movement and spread of cholera in the early onset of the epidemic was closely linked to proximity with the Artibonite River.
It had been reported that Kathmandu, the capital of Nepal, where the troops trained shortly before being stationed to Haiti, experienced a cholera outbreak on September 23 . The first batch of troops arrived in Haiti on October 8 , and the first cholera case was reported on October 12  (Figure 2B). Because none of the troops apparently exhibited symptoms of cholera during the pre-deployment medical examination, the MINUSTAH chief medical officer later revealed that no follow-up tests were done . However, the absence of symptoms did not prove that the troops were V. cholerae-free, as they could have been infected in the days following the medical examination and prior to deployment, or they could have been asymptomatic carriers , . Unfortunately, other than that done by the MINUSTAH, no independent testing was done of the troops to confirm the presence or absence of V. cholerae.
Comparative Genomics Traced a Single Source for the Epidemic
The first molecular study on the origin of V. cholerae in Haiti was published on December 9, 2010 . Chin and colleagues sequenced the complete genomes of two Haitian strains obtained from the outbreak, as well as epidemic strains from South America and Bangladesh, and compared them to those of epidemic-associated strains available in public databases. Comparison of single-nucleotide variations and hypervariable chromosomal elements in the genomes showed both of these Haitian strains to be genetically identical. While this is a small sample size, it was consistent with a clonal source for the outbreak. In addition, the study was able to genotype the two strains at polymorphic loci previously used for population genetic studies of V. cholerae, and this subtype had been previously observed in a broad region that included South Asia, Thailand, and Africa, but not the Americas. The study by Chin and colleagues suggested that cholera was introduced into Haiti through human transmission from a distant geographic source, most probably from South Asia (i.e., Bangladesh), although their conclusions were based upon a very limited strain analysis from both Haitian and global populations.
Two subsequent and larger genomic studies used 23  and 154  whole genome sequences to document the repeated historical spread of V. cholerae O1 from South Asia. These studies used up to nine more Haitian isolates and placed them into the context of the expanded strain genome collection. They found phylogenetic affinity between the 2010 Haitian strains and those seen in previous years from Cameroon, Bangladesh, India, and Pakistan. The Haitian isolates were nearly identical and again consistent with a single clonal outbreak. Contemporary (i.e., 2010) V. cholerae strains from Nepal were not included in these studies and the genomic association between Haitian and Nepalese V. cholerae was not differentiated from other South Asian or even African locations.
The first study to include strains from Nepal was published by Hendriksen and colleagues on August 23, 2011 . It compared the genomes of 24 strains isolated from five geographic regions in Nepal (between July 30 and November 1, 2010) with ten genomes of previously sequenced V. cholerae, including three from Haiti. All strains from Nepal, Haiti, and Bangladesh clustered together in a single monophyletic group (i.e., they shared a common ancestor). More importantly, the three Haitian and three Nepalese strains formed a very tight subgroup within the cluster, and these were almost identical, with only one or two nucleotide difference(s) in their core genome. This study, coupled with classical epidemiology , , showed convincing evidence that cholera was introduced into Haiti from an external source, with Nepal being the most likely origin.
Despite such strong evidence supporting the human transmission hypothesis, some scientists still stood by the climatic hypothesis. A study published on June 18, 2012 by Hasan and colleagues suggested that indigenous non-O1/O139 Haitian strains were involved in the outbreak . The study entailed the identification and comparison of V. cholerae from 81 stool samples taken from the beginning of the outbreak by traditional methods and comparative genomics. V. cholerae O1 was found in 48% of the samples, but more surprisingly, non-O1/O139 were identified in 21% of the samples. In addition, both O1 and non-O1/O139 strains were co-cultured in 7% of the samples, suggesting that non-O1/O139 strains may have played a role in the epidemic, whether alone or in concert with O1 strains. The authors stated that the assignment of attribution for cholera in Haiti remains controversial. However, scientists and Haitian public health officials supporting the human transmission hypothesis criticized the work, pointing out the unreliability of sampling methods  and that the study did not offer evidence that non-O1/O139 played a notable role in the epidemic .
A recent study led by scientists from the CDC and published on July 2, 2013 provided additional strong evidence to refute the climatic hypothesis . Katz and colleagues sequenced the genomes of V. cholerae strains isolated from different time points within a two-year period since the start of the outbreak. The genomic affinity of the Haitian and Nepalese strains was reaffirmed; they were clearly distinct from isolates circulating elsewhere in the world, and there was no evidence of novel gene acquisition by horizontal gene transfer. A molecular clock was calculated, and the date of the most recent common ancestor (MRCA) was estimated to be between July 23 to October 17, 2010 (with a 95% range credibility). The human transmission hypothesis suggesting Nepalese origins would stipulate that the MRCA was in Nepal prior to deployment of the MINUSTAH troops. Additionally, this time interval encompasses the reported cholera outbreak in Nepal (September 23) , the arrival of the Nepalese soldiers in Haiti (October 8) , and the first reported cholera cases (October 12 and 17) , , supporting the time frame of the outbreak proposed by previous studies (Figure 2B) , .
Whole Genome Sequencing as a Tool for Molecular Epidemiology
The investigations of the cholera outbreak in Haiti illustrated how traditional epidemiological investigations can be greatly enhanced by genomic sequencing and phylogenetic analysis. In this case, the analysis of hospital case records established a spatiotemporal pattern to the outbreak but failed to differentiate between the two competing hypotheses. The subsequent genomic analyses provided very strong evidence to support the human transmission hypothesis; thus, the climatic hypothesis could be rejected. These analyses, because of their ability to detect minute differences between different strains, also allowed the determination of the exact source of the outbreak. This can help enormously with prevention of future outbreaks and can also have legal implications. In a recent development, a lawsuit has been filed against the United Nations in the United States Federal Court for damages caused by the cholera outbreak . With the potential for legal action, genomic analysis methods have to be very rigorous, as the judicial system will require high standards to accept them as evidence.
In retrospect, the identification of the origin of cholera in Haiti was limited by two essential factors. First was that genome sequencing was used only in the later steps of the investigation, not as a “first responder” screening method for identification of infectious agents. We are clearly entering an era where genomic or even metagenomic screening will become a part of medical diagnostics. The second limitation was the absence of a public database containing sufficient genome sequences of recurring pathogens from various geographical locations. These two elements are not out of reach, and major efforts are underway to remove both limitations . For example, pipelines using current technologies to fully sequence a genome and perform key analyses for typing and identification within 24 hours are now becoming available . With the price of sequencing declining, comprehensive, geographically-informed genome sequence databases of pathogens could soon be a reality.
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