Whole Genome Sequencing and Spatial Analysis Identifies Recent Tuberculosis Transmission Hotspots in Ghana

Abstract

Whole genome sequencing (WGS) is progressively being used to investigate the transmission dynamics of Mycobacterium tuberculosis complex (MTBC). We used WGS analysis to resolve traditional genotype clusters and explored the spatial distribution of confirmed recent transmission clusters. Bacterial genomes from a total of 452 MTBC isolates belonging to large traditional clusters from a population-based study spanning July 2012 and December 2015 were obtained through short read next-generation sequencing using the illumina HiSeq2500 platform. We performed clustering and spatial analysis using specified R packages and ArcGIS. Of the 452 traditional genotype clustered genomes, 314 (69.5%) were confirmed clusters with a median cluster size of 7.5 genomes and an interquartile range of 4-12. Recent tuberculosis (TB) transmission was estimated as 24.7%. We confirmed the wide spread of a Cameroon sub-lineage clone with a cluster size of 78 genomes predominantly from the Ablekuma sub-district of Accra metropolis. More importantly, we identified a recent transmission cluster associated with isoniazid resistance belonging to the Ghana sub-lineage of lineage 4. WGS was useful in detecting unsuspected outbreaks; hence, we recommend its use not only as a research tool but as a surveillance tool to aid in providing the necessary guided steps to track, monitor, and control TB.

Keywords: Mycobacterium africanum; Mycobacterium tuberculosis; cluster; molecular epidemiology; recent transmission; whole genome sequence.

Figure 1

Pipeline for sample selection and whole genome sequencing. MTBC, Mycobacterium tuberculosis complex; WGS, Whole genome sequencing. ^aWe defined traditional genotyping clusters as previously described clusters identified using mycobacterial interspersed repetitive-unit–variable-number tandem-repeat analysis and spoligotyping tools. ^bGenomes with heterozygous SNPs >120 were classified as possible mixed infection or contamination and hence removed from further analysis.

Figure 2

Phylogenetic relationship of six M. tuberculosis complex longitudinal isolates obtained from three participants. The two major branches constitute the top two MTBC lineages found in Ghana and color coded as red for lineage 4 and brown for lineage 5. Lineage 4 is further split into the dominant Cameroon and Ghana sub-lineage. The sub-lineages, days between sampling for each participant as well as the number of SNPs observed between sample pairs are annotated on the tree rooted on M. canettii. This analysis involved seven nucleotide sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There was a total of 2104 positions in the final dataset. Evolutionary analyses were conducted in MEGA v10.0.5 (32).

Figure 3

Phylogenetic reconstruction of 452 M. tuberculosis complex isolates showing clustering at a threshold of 10 SNPs. The tree was built with an alignment file containing 11,041 variable positions and rooted on M. canettii. Black bars plotted on the tips of the branches indicate the clustered cases at the defined threshold of 10 SNPs. Blue bars represent large clusters (cluster size >5) with the number of clustered cases indicated in brackets. The three major branches constitute the three main MTBC lineages found in Ghana and color coded as red for lineage 4, brown for lineage 5, and green for lineage 6.

Figure 4

Clustering analysis stratified by cluster definition. (A) Frequency of clusters per cluster definition. (B) Estimated clustering rate per cluster definition. The estimated population size (N = 1000) used for estimating the clustering rate are explained in Materials and methods (see Supplementary Data). Singletons were calculated from the estimated population size of 1,000 individuals. IQR, interquartile range.

Figure 5

Phylogenetic reconstruction of 146 M. tuberculosis complex isolates rooted on M. canettii showing characteristics of the eight identified large clusters as defined by a threshold of 10 SNPs. The heat map shows some characteristics of the clustered cases including sampling period (column 1), gender (column 2), residential district (column 3), smear results (column 4), and drug resistance status to isoniazid (column 5). There was only one rifampicin resistant isolate (black arrow). The color codes are defined in the key. All cases belong to lineage 4.

Figure 6

Spatial distribution of pulmonary tuberculosis cases belonging to large clusters in and around Accra metropolis. The geographical distribution of cases from all eight large clusters is shown in (A) and the relative distribution of cases from the largest cluster highlighted in (B) stratified by Accra metropolis sub-district. Abbreviations: Able, Ablekuma sub-district; Okai, Okaikoi sub-district; AshK, AShiedu Keteke sub-district; OsuK, Osu Klottey sub-district; Ayaw, Ayawaso sub-district; Kpe, Kpeshie sub-district.