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

doi:10.1016/S1473-3099(16)30418-2

The Lancet Infectious Diseases

Volume 17, Issue 3, March 2017, Pages 275-284

https://doi.org/10.1016/S1473-3099(16)30418-2 Get rights and content

Summary

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.

Introduction

The worldwide emergence of multidrug-resistant (MDR) tuberculosis threatens the global eradication of tuberculosis. An estimated 480 000 cases of MDR tuberculosis occurred worldwide in 2015, but only one in ten cases were diagnosed, treated, and cured.¹ As well as acquisition of MDR tuberculosis during treatment for tuberculosis, person-to-person transmission of MDR strains occurs and can be increased by delayed diagnosis, prolonged treatment, and unfavourable treatment outcomes in patients with MDR tuberculosis. Therefore, understanding the causes and transmission patterns of MDR tuberculosis is crucial to inform public health efforts to reduce this disease.²

Molecular epidemiological methods that combine epidemiological investigations and genotyping of Mycobacterium tuberculosis strains provide the means to assess the recent transmission (within 2–3 years) of M tuberculosis and risk factors for transmission.³ However, traditional genotyping methods have limited discriminatory power and are limited in assessing homoplasy, which reduces their accuracy in identifying recent transmissions of M tuberculosis.⁴ High-throughput whole-genome sequencing provides increased resolution and accuracy over older methods, and is a powerful tool to study the transmission of M tuberculosis.4, 5, 6, 7, 8, 9, 10 Furthermore, phylogenetic networks based on whole-genome sequencing can be used to identify putative source cases, super-spreaders, and transmission directions in the absence of, or complementary to, extensive epidemiological data.8, 9 However, few studies have applied whole-genome sequencing to address recent transmission of MDR tuberculosis at the population level.5, 10

China has a high prevalence of drug-resistant tuberculosis and the second largest number of MDR cases worldwide.¹ The high risk of incident MDR tuberculosis among patients who have previously been treated for tuberculosis has resulted in a common belief that most cases of MDR tuberculosis are due to acquired resistance, and led to the allocation of resources to improve treatment. However, many cases of MDR tuberculosis are due to transmission of MDR strains,11, 12, 13, 14 but few studies provide direct evidence of transmission of MDR tuberculosis at the population level. Thus, we hypothesised that the transmission of MDR strains has a major role in the high prevalence of MDR tuberculosis in China. We did a population-based retrospective study in Shanghai, China, using whole-genome sequencing and epidemiological investigations. We quantified the magnitude of MDR tuberculosis arising from the transmission of MDR strains, tracked transmission patterns, and identified the risk factors for transmission.

Research in context

Evidence before the study

We searched PubMed for molecular epidemiology studies of Mycobacterium tuberculosis that used whole-genome sequencing using the search terms “tuberculosis”, “multidrug resistance”, “whole genome sequencing”, and “transmission”. We only selected articles published in English before Oct 31, 2016. We identified 20 studies published from 2011 in which whole-genome sequencing was used to investigate transmission of M tuberculosis at the population level. We also searched the China Knowledge Resource Integrated Database, with the same search terms, for relevant papers published in Chinese, but did not identify any studies. In 2011, Gardy and colleagues applied whole-genome sequencing-based analysis to a large mycobacterial interspersed-repetitive-unit–variable-number tandem-repeat cluster of M tuberculosis and showed that whole-genome sequencing had higher resolution compared with traditional genotyping. Walker and colleagues evaluated the potential of whole-genome sequencing to delineate outbreaks of M tuberculosis and to measure the recent transmission of M tuberculosis in the UK. In a large-scale population-based study using whole-genome sequencing, Guerra-Assunção and colleagues showed that most cases of tuberculosis in a high-incidence setting in Malawi were caused by just one lineage of M tuberculosis. Casali and colleagues investigated the long-term evolution and endemic spread of MDR tuberculosis in a Russian population, and an additional study of an outbreak of isonazid-resistant tuberculosis in London identified a cluster of multidrug-resistant (MDR) strains, which involved rare rifampin-resistant mutations.

Added value of this study

Although several studies used whole-genome sequencing to track transmission of M tuberculosis, few studies used this approach to address transmission of MDR tuberculosis at the population level. To our knowledge, we describe the first population-based study combining genomics with detailed epidemiological data to identify transmission pathways of MDR tuberculosis in a region over time in China, the country with the second highest number of MDR tuberculosis cases in the world. We provide direct evidence of the emergence of diversity in M tuberculosis subpopulations within hosts and the fixation of specific subpopulations of M tuberculosis between hosts along a chain of transmission. Meanwhile, many MDR strains became more resistant during the recent transmission. Inadequate treatment was considered to be the most common way of developing MDR tuberculosis; however, our data show that most of the patients in our study population who had MDR tuberculosis had been infected with MDR strains. We also found that a majority of transmission events occurred in settings such as residential communities or complexes and related public facilities.

Implications of all the available evidence

Whole-genome sequencing provides greater precision than traditional genotyping to study recent transmission of MDR strains of M tuberculosis. Whole-genome sequencing is also useful to detect heterogeneity of strain diversity within and between hosts, which can be used to infer the transmission trajectory of MDR strains. Our findings suggest that strategies and interventions to halt ongoing transmission of MDR strains should be a priority for tuberculosis control programmes in China and other settings with a high burden of MDR tuberculosis.

Section snippets

Study design and population

Shanghai is the most populous city in China, with an estimated population of 24 million, and has a reasonably well-functioning tuberculosis control programme. All patients with suspected pulmonary tuberculosis are referred to local designated hospitals, where the diagnosis is made by sputum smear and culture. Through a routine surveillance system, all cases of tuberculosis are reported to Shanghai Municipal Centre for Disease Control and Prevention (CDC).

This population-based, retrospective

Results

7982 patients tested positive for M tuberculosis and had their isolates sent for drug susceptibility testing during the study (figure 1). Of the 324 patients with MDR tuberculosis who had a clinical isolate suitable for analysis (figure 1; appendix p 10), 191 (59%) were treatment-naive, 238 (73%) were male, and the median age was 39 years (range 16–88).

VNTR genotyping analysis showed that 125 (39%) of 324 patients with MDR tuberculosis had isolates with genotypic patterns that were identical to

Discussion

Our strategy combined traditional genotyping, whole-genome sequencing, and epidemiological investigation. Transmission of MDR strains accounted for most (73%) cases of MDR tuberculosis overall. Residential communities or complexes, and related public facilities, were the most common transmission settings. Whole-genome sequencing analyses were highly informative to determine the transmission patterns of MDR strains and the ongoing acquisition of additional antibiotic resistance during

References (31)

T Luo et al.
Whole-genome sequencing to detect recent transmission of Mycobacterium tuberculosis in settings with a high burden of tuberculosis
Tuberculosis (Edinb)
(2014)
TM Walker et al.
Whole-genome sequencing to delineate Mycobacterium tuberculosis outbreaks: a retrospective observational study
Lancet Infect Dis
(2013)
TM Walker et al.
Assessment of Mycobacterium tuberculosis transmission in Oxfordshire, UK, 2007–12, with whole pathogen genome sequences: an observational study
Lancet Respir Med
(2014)
LF Anderson et al.
Transmission of multidrug-resistant tuberculosis in the UK: a cross-sectional molecular and epidemiological study of clustering and contact tracing
Lancet Infect Dis
(2014)
PK Moonan et al.
Transmission of multidrug-resistant tuberculosis in the USA: a cross-sectional study
Lancet Infect Dis
(2013)
EA Kendall et al.
Burden of transmitted multidrug resistance in epidemics of tuberculosis: a transmission modelling analysis
Lancet Respir Med
(2015)
JM Bryant et al.
Whole-genome sequencing to establish relapse or re-infection with Mycobacterium tuberculosis: a retrospective observational study
Lancet Respir Med
(2013)
Global tuberculosis report 2016
E Nathanson et al.
MDR tuberculosis—critical steps for prevention and control
N Engl J Med
(2010)
PF Barnes et al.
Molecular epidemiology of tuberculosis
N Engl J Med
(2003)

JL Gardy et al.

Whole-genome sequencing and social-network analysis of a tuberculosis outbreak

N Engl J Med

(2011)

N Casali et al.

Evolution and transmission of drug-resistant tuberculosis in a Russian population

Nat Genet

(2014)

KA Cohen et al.

Evolution of extensively drug-resistant tuberculosis over four decades: whole genome sequencing and dating analysis of Mycobacterium tuberculosis isolates from KwaZulu-Natal

PLoS Med

(2015)

N Casali et al.

Whole genome sequence analysis of a large isoniazid-resistant tuberculosis outbreak in London: a retrospective observational study

PLoS Med

(2016)

CC Dobler et al.

Multidrug-resistant tuberculosis in patients for whom first-line treatment failed, Mongolia, 2010-2011

Emerg Infect Dis

(2015)

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A population-based retrospective study was conducted using whole-genome sequencing (WGS) to determine the characteristics of drug resistance and the transmission patterns.
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Our results suggest that drug-resistant strains are more transmissible than susceptible strains and that transmission is the major driving force of the current DR-TB epidemic in Kashgar.
The epidemiology, transmission, diagnosis, and management of drug-resistant tuberculosis—lessons from the South African experience
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The emerging threat of fluroquinolone-, bedaquiline-, and linezolid-resistant Mycobacterium tuberculosis in China: Observations on surveillance data
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Drug-resistant tuberculosis (TB), especially multidrug-resistant tuberculosis (MDR-TB), constitutes a major obstacle to fulfill end TB strategy globally. Although fluoroquinolones (FQs), linezolid (LZD) and bedaquiline (BDQ) were classified as Group A drugs for MDR-TB treatment, our knowledge of the prevalence of TB which were resistant to Group A drugs in China is quite limited.
In this study, we conducted a prospective multicenter surveillance study in China to determine the proportion of TB patients that were resistant to Group A drugs. A total of 1877 TB patients were enrolled from 2022 at four TB specialized hospitals. The drug susceptibility of isolated strains was conducted using the MGIT 960 system and the molecular mechanisms conferring drug resistance were investigated by Sanger sequencing.
12.9% of isolates were resistant to levofloxacin (LFX), 13.2% were resistant to moxifloxacin (MOX), 0.2% were resistant to bedaquiline (BDQ), and 0.8% were resistant to linezolid (LZD). Totally, 14.0% and 0.4% were classified as multidrug resistant- (MDR-) and extensively drug resistant- (XDR-) TB. The drug resistance was more common in retreated TB cases compared to new cases. In addition, 70.0% of fluoroquinolone (FQ)-resistant isolates harbored mutations in the gyrA and gyrB gene. By contrast, the common drug-resistant mutations were only found in 50% BDQ-resistant and 20% LZD-resistant isolates.
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With improved tuberculosis (TB) control programs, the incidence of TB in China declined dramatically over the past few decades, but recently the rate of decrease has slowed, especially in large cities such as Shanghai. To help formulate strategies to further reduce TB incidence, we performed a 10-year study in Songjiang, a district of Shanghai, to delineate the characteristics, transmission patterns, and dynamic changes of the local TB burden.
We conducted a population-based study of culture-positive pulmonary TB patients diagnosed in Songjiang during 2011–2020. Genomic clusters were defined with a threshold distance of 12-single-nucleotide-polymorphisms based on whole-genome sequencing, and risk factors for clustering were identified by logistic regression. Transmission inference was performed using phybreak. The distances between the residences of patients were compared to the genomic distances of their isolates. Spatial patient hotspots were defined with kernel density estimation.
Of 2212 enrolled patients, 74.7% (1652/2212) were internal migrants. The clustering rate (25.2%, 558/2212) and spatial concentrations of clustered and unclustered patients were unchanged over the study period. Migrants had significantly higher TB rates but less clustering than residents. Clustering was highest in male migrants, younger patients and both residents and migrants employed in physical labor. Only 22.1% of transmission events occurred between residents and migrants, with residents more likely to transmit to migrants. The clustering risk decreased rapidly with increasing distances between patient residences, but more than half of clustered patient pairs lived ≥5 km apart. Epidemiologic links were identified for only 15.6% of clustered patients, mostly in close contacts.
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Shanghai Municipal Science and Technology Major Project (ZD2021CY001), National Natural Science Foundation of China (82272376), National Research Council of Science and Technology Major Project of China (2017ZX10201302-006).
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2023, Journal of Infection and Public Health
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A total of 95 EDR-TB patients were included in our analysis. Up on the WGS based genotyping, 94 (94/95, 98.9%) out of 95 were identified as lineage 2 (East Asia). The pairwise genomic distance analysis identified 7 clusters, ranging in size from 2 to 5 isolates. The clustering rate of EDR-TB was 21.1%; while no patients had significantly higher odds of clustering. All isolates harbor rpoB RRDR mutations that confer RIF resistance and katG or inhA promoter mutations that confer INH resistance. Of 95 EDR-TB isolates, a total of 15 mutation types were recorded in the transcriptional regulator mmpR5. In vitro susceptibility testing results revealed that 14 (14/15, 93.3%) out of 15 mutation types were resistant to CFZ; whereas only 3 (3/15, 20.0%) showed resistance to BDQ. Interestingly, 12 isolates harbored mutations within rrl locus, whereas only mutations at positions 2294 and 2296 conferred CLA resistance. Favorable outcomes of EDR-TB patients were positively associated with more effective drugs in the regimes.
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Effect of compensatory evolution in the emergence and transmission of rifampicin-resistant Mycobacterium tuberculosis in Cape Town, South Africa: a genomic epidemiology study
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Experimental data show that drug-resistance-conferring mutations are often associated with a decrease in the replicative fitness of bacteria in vitro, and that this fitness cost can be mitigated by compensatory mutations; however, the role of compensatory evolution in clinical settings is less clear. We assessed whether compensatory evolution was associated with increased transmission of rifampicin-resistant tuberculosis in Khayelitsha, Cape Town, South Africa.
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Between Jan 1, 2008, and Dec 31, 2017, 2161 individuals were diagnosed with multidrug-resistant or rifampicin-resistant tuberculosis in Khayelitsha, Cape Town, South Africa. Whole-genome sequences were available for 1168 (54%) unique individual M tuberculosis isolates. Compensatory evolution was associated with smear-positive pulmonary disease (adjusted odds ratio 1·49, 95% CI 1·08–2·06) and a higher number of drug-resistance-conferring mutations (incidence rate ratio 1·38, 95% CI 1·28–1·48). Compensatory evolution was also associated with increased transmission of rifampicin-resistant disease between individuals (adjusted odds ratio 1·55; 95% CI 1·13–2·12), independent of other patient and bacterial factors.
Our findings suggest that compensatory evolution enhances the in vivo fitness of drug-resistant M tuberculosis genotypes, both within and between patients, and that the in vitro replicative fitness of rifampicin-resistant M tuberculosis measured in the laboratory correlates with the bacterial fitness measured in clinical settings. These results emphasise the importance of enhancing surveillance and monitoring efforts to prevent the emergence of highly transmissible clones capable of rapidly accumulating new drug resistance mutations. This concern becomes especially crucial at present, because treatment regimens incorporating novel drugs are being implemented.
Funding for this study was provided by a Swiss and South Africa joint research award (grant numbers 310030_188888, CRSII5_177163, and IZLSZ3_170834), the European Research Council (grant number 883582), and a Wellcome Trust fellowship (to HC; reference number 099818/Z/12/Z). ZS-D was funded through a PhD scholarship from the South African National Research Foundation and RMW was funded through the South African Medical Research Council.

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^†: Contributed equally

^‡: Contributed equally

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

Summary

Background

Methods

Findings

Interpretation

Funding

Introduction

Section snippets

Study design and population

Results

Discussion

Tuberculosis (Edinb)

Lancet Infect Dis

Lancet Respir Med

Lancet Infect Dis

Lancet Infect Dis

Lancet Respir Med

Lancet Respir Med

Global tuberculosis report 2016

MDR tuberculosis—critical steps for prevention and control

N Engl J Med

Molecular epidemiology of tuberculosis

N Engl J Med

Whole-genome sequencing and social-network analysis of a tuberculosis outbreak

N Engl J Med

Evolution and transmission of drug-resistant tuberculosis in a Russian population

Nat Genet

Evolution of extensively drug-resistant tuberculosis over four decades: whole genome sequencing and dating analysis of Mycobacterium tuberculosis isolates from KwaZulu-Natal

PLoS Med

Whole genome sequence analysis of a large isoniazid-resistant tuberculosis outbreak in London: a retrospective observational study

PLoS Med

Multidrug-resistant tuberculosis in patients for whom first-line treatment failed, Mongolia, 2010-2011

Emerg Infect Dis

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