The Molecular and Spatial Epidemiology of Typhoid Fever in Rural Cambodia

Abstract

Typhoid fever, caused by the bacterium Salmonella Typhi, is an endemic cause of febrile disease in Cambodia. The aim of this study was to better understand the epidemiology of pediatric typhoid fever in Cambodia. We accessed routine blood culture data from Angkor Hospital for Children (AHC) in Siem Reap province between 2007 and 2014, and performed whole genome sequencing (WGS) on the isolated bacteria to characterize the S. Typhi population. The resulting phylogenetic information was combined with conventional epidemiological approaches to investigate the spatiotemporal distribution of S. Typhi and population-level risk factors for reported disease. During the study period, there were 262 cases of typhoid within a 100 km radius of AHC, with a median patient age of 8.2 years (IQR: 5.1-11.5 years). The majority of infections occurred during the rainy season, and commune incidences as high as 11.36/1,000 in children aged <15 years were observed over the study period. A population-based risk factor analysis found that access to water within households and increasing distance from Tonle Sap Lake were protective. Spatial mapping and WGS provided additional resolution for these findings, and confirmed that proximity to the lake was associated with discrete spatiotemporal disease clusters. We confirmed the dominance of MDR H58 S. Typhi in this population, and found substantial evidence of diversification (at least seven sublineages) within this single lineage. We conclude that there is a substantial burden of pediatric typhoid fever in rural communes in Cambodia. Our data provide a platform for additional population-based typhoid fever studies in this location, and suggest that this would be a suitable setting in which to introduce a school-based vaccination programme with Vi conjugate vaccines.

Fig 1. The annual and seasonal distribution of typhoid fever cases at Angkor Hospital for Children in Cambodia.

a) The annual number of culture confirmed (solid line) and non-confirmed (broken line) typhoid cases at AHC from 2007 to 2014. b) The annual number of total admissions at AHC from 2007 to 2014. c) The mean monthly count of typhoid cases aggregated from 2007 to 2014. d) The average monthly rainfall (mm) per month over the study period.

Fig 2. The spatial distribution of typhoid fever cases in Siem Reap province, Cambodia.

a) North oriented map of Cambodia, the black cross shows the location of AHC. b) Map showing the population density (people/km², color-coding in key) of the 78 communes within the typhoid study area. AHC is shown by the black cross, the black border denotes Siem Reap province and the left and right asterisks are mark the locations of the communes with highest incidence of typhoid fever; Kaoh Chiveang and Kampong Kleang, respectively. c) Map of the study area showing the rate of reported typhoid cases per 1,000 population under the age of 15 years (color-coding in key). d) Map of the study area showing significant spatiotemporal clusters of typhoid during the study period, the size of the grey circles corresponds to the radius of the cluster and the years of the clusters are denoted.

Fig 3. The phylogenetic structure of the H58 lineage of Cambodian Salmonella Typhi.

a) Maximum likelihood phylogenetic tree of the 203 H58 isolates identified during this project (scale bar denotes SNP differences). The sub-lineages are shown on the major branches. Isolates exhibiting a multi-drug resistance (MDR) phenotype are indicated by black nodes. The tree is midpoint-rooted for the purpose of clarity. Bootstrap values >85% are indicated by an asterisk. b) Minimum spanning tree subdividing H58 lineage III and IV into the various sublineages (IIIa, IIIb, IIIc, IV, IVa, IVb, IVc). The various sublineages are color-coded for reference and the number of each variant is indicated by the cluster size. The number on each of the branches signifies the number of SNPs between each cluster.

Fig 4. The spatiotemporal distribution of the various Salmonella Typhi lineages/sublineages in Siem Reap province, Cambodia.

a) Bar chart shows the annual distribution of the various S. Typhi lineages/sublineages from 2007 to 2012; sublineages are color-coded as in Fig 3b. b) Maps showing significant spatiotemporal clusters identified for sublineages IIIc, IV, IVb and IVc. The timing of each cluster is shown by the year in black text and the dotted circle represents the radius of the detected cluster. Background colors represent the rate of each sublineage per 1,000 population aged under 15 years. The incidence rates vary between sublineages, ranging from 0 to a maximum of 0.8 (IIIc), 3.12 (IV), 2.56 (IVb) and 5.84 (IVc) 5.84 cases/1,000 population aged under 15 years.