Population Genomics of Mycobacterium leprae Reveals a New Genotype in Madagascar and the Comoros

Abstract

Human settlement of Madagascar traces back to the beginning of the first millennium with the arrival of Austronesians from Southeast Asia, followed by migrations from Africa and the Middle East. Remains of these different cultural, genetic, and linguistic legacies are still present in Madagascar and other islands of the Indian Ocean. The close relationship between human migration and the introduction and spread of infectious diseases, a well-documented phenomenon, is particularly evident for the causative agent of leprosy, Mycobacterium leprae. In this study, we used whole-genome sequencing (WGS) and molecular dating to characterize the genetic background and retrace the origin of the M. leprae strains circulating in Madagascar (n = 30) and the Comoros (n = 3), two islands where leprosy is still considered a public health problem and monitored as part of a drug resistance surveillance program. Most M. leprae strains (97%) from Madagascar and Comoros belonged to a new genotype as part of branch 1, closely related to single nucleotide polymorphism (SNP) type 1D, named 1D-Malagasy. Other strains belonged to the genotype 1A (3%). We sequenced 39 strains from nine other countries, which, together with previously published genomes, amounted to 242 genomes that were used for molecular dating. Specific SNP markers for the new 1D-Malagasy genotype were used to screen samples from 11 countries and revealed this genotype to be restricted to Madagascar, with the sole exception being a strain from Malawi. The overall analysis thus ruled out a possible introduction of leprosy by the Austronesian settlers and suggests a later origin from East Africa, the Middle East, or South Asia.

Keywords: Comoros; Madagascar; Mycobacterium leprae; genomics; leprosy; phylogeography.

FIGURE 1

Sampling sites in Madagascar and the Comoros. Pie charts indicate the regions where patients originated and are color-coded based on PCR and genotyping results, as indicated in the caption box. Numbers within circles represent different patients tested when there is more than one patient. Most of the samples were collected in Antananarivo State. Boxed circles refer to the eight patients of unknown location in the island. Data used for the map are available in Supplementary Tables S1, S2 (86 patients). Multiple samples derived from one patient are counted only once. The figure was drawn in Inkscape (Yuan et al., 2016). The map was downloaded from https://www.amcharts.com/svg-maps/ under a free license and modified for the current figure.

FIGURE 2

Phylogeography of Mycobacterium leprae strains. (A) Maximum parsimony tree of 241 genomes of M. leprae representing the nine branches and the 16 genotypes. Support values were obtained by bootstrapping 500 replicates. Branch lengths are proportional to nucleotide substitutions. The tree is rooted using Mycobacterium lepromatosis. The 1D-Malagasy genotype, discovered in this investigation, is shown in blue. Newly sequenced genomes are shown in red. (B) Zoom into branch 1 (genotypes 1A, 1B, 1D, and the 1D-Malagasy) and 2E of the maximum parsimony tree from (A). The 1D-Malagasy genotype is indicated with the dotted blue line and the strain from Malawi in bold red. (C) Global distribution of the genotypes from the branches 1 and 2E. Genotypes are colored as in (B). Strains from the canonical 1D are found in 12 countries, while the 1D-Malagasy is found only in Madagascar, the Comoros, and Malawi. The arrows indicate possible routes of leprosy introduction into Madagascar and Comoros with the estimated time frame.