Transmission of multidrug-resistant Mycobacterium tuberculosis in Shanghai, China: a retrospective observational study using whole-genome sequencing and epidemiological investigation

Abstract

Background: Multidrug-resistance is a substantial threat to global elimination of tuberculosis. Understanding transmission patterns is crucial for control of the disease. We used a genomic and epidemiological approach to assess recent transmission of multidrug-resistant (MDR) tuberculosis and identify potential risk factors for transmission.

Methods: We did a population-based, retrospective study of patients who tested positive for tuberculosis between Jan 1, 2009, and Dec 31, 2012, in Shanghai, China. We did variable-number-of-tandem-repeat genotyping and whole-genome sequencing of isolates. We measured strain diversity within and between genomically clustered isolates. Genomic and epidemiological data were combined to construct transmission networks.

Findings: 367 (5%) of 7982 patients with tuberculosis had MDR tuberculosis and 324 (88%) of these had isolates available for genomic analysis. 103 (32%) of the 324 MDR strains were in 38 genomic clusters that differed by 12 or fewer single nucleotide polymorphisms (SNPs), indicating recent transmission of MDR strains. Patients who had delayed diagnosis or were older than 45 years had high risk of recent transmission. 235 (73%) patients with MDR tuberculosis probably had transmission of MDR strains. Transmission network analysis showed that 33 (87%) of the 38 clusters accumulated additional drug-resistance mutations through emergence or fixation of mutations during transmission. 68 (66%) of 103 clustered MDR strains had compensatory mutations of rifampicin resistance.

Interpretation: Recent transmission of MDR tuberculosis strains, with increasing drug-resistance, drives the MDR tuberculosis epidemic in Shanghai, China. Whole-genome sequencing can measure of the heterogeneity of drug-resistant mutations within and between hosts and help to determine the transmission patterns of MDR tuberculosis.

Funding: National Science and Technology Major Project, National Natural Science Foundation of China, and US National Insitutes of Health.

Figure 1

Classification of multidrug-resistant tuberculosis based on treatment history and genomic analysis. PTB, pulmonary tuberculosis; DST, drug susceptibility testing; MDR-TB, multidrug-resistant tuberculosis.

Figure 2

Distribution of the number of single nucleotide polymorphisms (SNPs) in isolates from the closest multidrug-resistant tuberculosis cases within a cluster. Epidemiological links were indicated (Black).

Figure 3

Maximum-likelihood whole-genome SNP-based phylogeny of multidrug-resistant isolates and mutations in RNA polymerase subunits rpoA, rpoB, and rpoC. Branches with colours indicate the sublineages: non-Beijing strains (yellow), “Ancient” Beijing strain (ice blue), “Modern” Beijing strain (sky blue); Square beside the nodes show the presence of pre-extensively drug resistant (pre-XDR, purple) and extensively drug resistant (XDR, orange) genotypes among genomically clustered MDR-TB strains. The amino-acid change caused by mutation of rpoABC is annotated along the periphery of the phylogeny.

Figure 4

Transmission networks of MDR-TB based on genetic distances and drug-resistance mutations. (A) The Median-joining (M-J) networks for four main MDR-TB clusters. For each network, the arrow indicates the root and the circles represent M. tuberculosis (Mtb) isolates (numbered according to patient identification). Mtb isolates were separated by lines with length (as suggested by the dots) representing genetic distance. Isolates with identical genomes were grouped in the same circle. The color of each circle represents multidrug-resistant (blue), pre-XDR (yellow), and XDR (pink) tuberculosis patients. The colored shapes on the branches or within the circles represent resistance mutations. (B) Matrix of resistance mutations for the clusters in panel A. Fixed mutations were highlighted with colors, heterogeneous mutations (simultaneous existence of mutant and wild-type alleles) have a white background and wild-type alleles are indicated as dots in a light-blue background. The heterogeneous mutations were mapped into corresponding circles in the M-J networks to indicate emerging resistance. The fixed mutations were mapped onto the branches by assuming parsimonious acquisition with no reversion. Different shapes of the same color were used to indicate different mutations for the same resistance phenotype within each network. Abbreviations: TB, tuberculosis; MDR, multi drug-resistant; XDR, extensively drug-resistant; INH, isoniazid; RIF, rifampin; SM, streptomycin; EMB, ethambutol; PZA, pyrazinamide; FLQ, fluoroquinolones; AMI, amikacin; RIFc=RIF compensatory.

Figure 5

Results of the detailed epidemiologic investigation and genomic analysis of cluster C-09. (A) Putative transmission network based on epidemiological links. (B) Time of onset of symptoms, diagnosis, and treatment. Based on Walker, T.M., et al. (C) Median-joining (M-J) network and drug-resistance mutations of clusters. Each circle represents clustered patients who were infected with strains separated by no SNPs; M. tuberculosis (Mtb) isolates were separated by lines with length (as suggested by the dots) representing genetic distance. The color of each circle represents multidrug-resistant (blue), pre-XDR (yellow) and XDR (pink) tuberculosis patients. The heterogeneous mutations were mapped into corresponding circles in the M-J networks to indicate emerging resistance. The fixed mutations were mapped onto the branches by assuming parsimonious acquisition with no reversion. Different shapes of the same color were used to indicate different mutations for the same resistance phenotype within each network.