Phylogeography and transmission of M. tuberculosis in Moldova: A prospective genomic analysis

Abstract

Background: The incidence of multidrug-resistant tuberculosis (MDR-TB) remains critically high in countries of the former Soviet Union, where >20% of new cases and >50% of previously treated cases have resistance to rifampin and isoniazid. Transmission of resistant strains, as opposed to resistance selected through inadequate treatment of drug-susceptible tuberculosis (TB), is the main driver of incident MDR-TB in these countries.

Methods and findings: We conducted a prospective, genomic analysis of all culture-positive TB cases diagnosed in 2018 and 2019 in the Republic of Moldova. We used phylogenetic methods to identify putative transmission clusters; spatial and demographic data were analyzed to further describe local transmission of Mycobacterium tuberculosis. Of 2,236 participants, 779 (36%) had MDR-TB, of whom 386 (50%) had never been treated previously for TB. Moreover, 92% of multidrug-resistant M. tuberculosis strains belonged to putative transmission clusters. Phylogenetic reconstruction identified 3 large clades that were comprised nearly uniformly of MDR-TB: 2 of these clades were of Beijing lineage, and 1 of Ural lineage, and each had additional distinct clade-specific second-line drug resistance mutations and geographic distributions. Spatial and temporal proximity between pairs of cases within a cluster was associated with greater genomic similarity. Our study lasted for only 2 years, a relatively short duration compared with the natural history of TB, and, thus, the ability to infer the full extent of transmission is limited.

Conclusions: The MDR-TB epidemic in Moldova is associated with the local transmission of multiple M. tuberculosis strains, including distinct clades of highly drug-resistant M. tuberculosis with varying geographic distributions and drug resistance profiles. This study demonstrates the role of comprehensive genomic surveillance for understanding the transmission of M. tuberculosis and highlights the urgency of interventions to interrupt transmission of highly drug-resistant M. tuberculosis.

Fig 1

(A) Map of culture-confirmed TB patients in Moldova. The center of each circle represents the geometric center of the localities/region (709 named localities within 50 regions) where the case was diagnosed and sampled. The scale indicates the number of culture-confirmed TB patients (n = 2,236). The Transnistrian region of Moldova is highlighted. The geographic distribution of the notified incidence of all culture-confirmed (B) TB and (C) MDR-TB by locality. The colors show the distribution of notified case per population and localities colored dark gray have missing population data. The map data were extracted from the GADM database (www.gadm.org/download_country.html). MDR-TB, multidrug-resistant tuberculosis; TB, tuberculosis.

Fig 2

(A) M–L phylogeny of 1,834 Moldova M. tuberculosis isolates based on 43,284 variable sites. The outer bands represent the in silico drug-resistant profiles, treatment history of participant and the region where the isolates were sampled from. The tree is rooted to Mycobacterium bovis (branch in green). L2 denotes lineage 2 (light orange) and L4 lineage 4 (light blue). Three major clades from the Ural/ lineage 4.2.1 (clade 1) and Beijing/lineage2.2.1 (clades 2 to 3) are shaded. The main nodes of the tree have 100% bootstrap support. (B) Phylogenetic distribution of resistance-related genotypes. The columns depict loci associated with drug resistance. “P” followed by a subscription of gene name indicates the promotor region. Colored bands of each column represent different polymorphisms. DR, drug resistance; MDR, multidrug resistance; MDR-TB, multidrug-resistant tuberculosis; M–L, maximum–likelihood.

Fig 3

(A–C) Tree visualizations for 3 large putative transmission clusters (N ≥ 10 isolates), each showing the location of cases in either the Moldova or Transnistria regions along with resistance/susceptibility to 12 anti-TB drugs, as identified by in silico prediction. (D, E) Spatial distribution of 3 largest clusters (Cluster 1, 2, and 7) in the Ural/Lineage 4.2.1 and Beijing/lineage 2.2.1 clades. The map data were extracted from the GADM database (www.gadm.org/download_country.html). MDR-TB, multidrug-resistant tuberculosis; TB, tuberculosis.

Fig 4

(A–C) Coalescent Bayesian Skyline plots of the 3 large clades among Ural/lineage 4.2.1 and Beijing/lineage 2.2.1 with specific resistant mutations (detailed in Fig 2B) using an uncorrelated log normal relaxed clock model. The 2 blue lines are the upper and lower bounds of the 95% HPD interval. The x-axis is the time in years and the y-axis is on a log scale. (D) Density distribution of within-clade pairwise SNPs distance of clades 1 to 3. HPD, highest posterior density; SNP, single nucleotide polymorphism.