MAJOR ARTICLE Transmission of Mycobacterium tuberculosis in China: A Population-Based Molecular Epidemiologic Study Chongguang Yang,1 Xin Shen,2 Ying Peng,3 Rushu Lan,4 Yuling Zhao,5 Bo Long,6 Tao Luo,1 Guomei Sun,1 Xia Li,1 Ke Qiao,1 Xiaohong Gui,2 Jie Wu,2 Jiying Xu,5 Fabin Li,3 Dingyue Li,6 Feiying Liu,4 Mei Shen,2 Jianjun Hong,7 Jian Mei,2,a Kathryn DeRiemer,8,a and Qian Gao1,a 1Key Laboratory of Medical Molecular Virology of Ministries of Education and Health, Institutes of Biomedical Sciences and Institute of Medical Microbiology, Shanghai Medical College, Fudan University, 2Department of Tuberculosis Control, Shanghai Municipal Center for Disease Control and Prevention, 3Tuberculosis Control Center of Heilongjiang Province, Harbin, 4Guangxi Zhuang Autonomous Region Center for Disease Control and Prevention, Nanning, 5Henan Center for Disease Control and Prevention, Zhengdong New District, Zhengzhou, 6Sichuan Center for Disease Control and Prevention, Chengdu, and 7Department of Tuberculosis Control, Songjiang District Center for Disease Control and Prevention, Shanghai, People’s Republic Downloaded from of China; and 8School of Medicine, University of California, Davis (See the Editorial Commentary by Arend and Soolingen on pages 228–32.) Background. Understanding the transmission of Mycobacterium tuberculosis is essential for the development of efficient tuberculosis control strategies. China has the second-largest tuberculosis burden in the world. Recent trans- http://cid.oxfordjournals.org/ mission and infection with M. tuberculosis, particularly drug-resistant strains, may account for many new tubercu- losis cases. Methods. We performed a population-based molecular epidemiologic study of pulmonary tuberculosis in China during1July2009to30June2012.Wedefined clusters as cases with identical variable number tandem repeat genotype patterns and identified the risk factors associated with clustering, by logistic regression. Relative transmis- sion rates were estimated by the sputum smear status and drug susceptibility status of tuberculosis patients. Results. Among 2274 culture-positive tuberculosis patients with genotyped isolates, there were 705 (31.0%) by guest on June 28, 2015 tuberculosis patients in 287 clusters. Multidrug-resistant (MDR) tuberculosis (adjusted odds ratio [aOR], 1.86; 95% confidence interval [CI], 1.25–2.63) and infection with a Beijing family strain (aOR, 1.56; 95% CI, 1.23– 2.96) were associated with clustering. Eighty-four of 280 (30.0%) clusters had a putative source case that was sputum smear negative, and 30.6% of their secondary cases were attributed to transmission by sputum smear–negative pa- tients. The relative transmission rate for sputum smear negative compared with sputum smear–positive patients was 0.89 (95% CI, .68–1.10), and was 1.51 (95% CI, 1.00–2.24) for MDR tuberculosis vs drug-susceptible tuberculosis. Conclusions. Recent transmission of M. tuberculosis, including MDR strains, contributes substantially to tuberculosis disease in China. Sputum smear–negative cases were responsible for at least 30% of the secondary cases. Interventions to reduce the transmission of M. tuberculosis should be implemented in China. Keywords. tuberculosis; molecular epidemiology; recent transmission; China. Received 27 October 2014; accepted 18 February 2015; electronically published Tuberculosis remains a major threat to global health, 31 March 2015. and is a leading cause of death in many developing cou- aJ. M., K. D., and Q. G. contributed equally to this work. Correspondence: Qian Gao, PhD, Key Laboratory of Medical Molecular Virology of ntries. Infection with Mycobacterium tuberculosis leads Ministries of Education and Health, Shanghai Medical College, Fudan University, to tuberculosis disease by 2 main mechanisms: infection 138 Yi Xue Yuan Road, Shanghai, China, 200032 ([email protected]). Clinical Infectious Diseases® 2015;61(2):219–27 and rapid progression to disease from a recent trans- © The Author 2015. Published by Oxford University Press on behalf of the Infectious mission event, and reactivation from latent tuberculosis Diseases Society of America. All rights reserved. For Permissions, please e-mail: [email protected]. due to a remote infection event and progression to dis- DOI: 10.1093/cid/civ255 ease [1–4].It is important to distinguish between these 2 Transmission of M. tuberculosis in China • CID 2015:61 (15 July) • 219 mechanisms of disease, because each requires different preven- were enrolled. The study protocol was approved by the institution- tion and control strategies. al review board of the Institutes of Biomedical Sciences (protocol The development of molecular epidemiology has allowed re- review No. 43), Fudan University. searchers to assess and quantify the recent transmission and re- activation of M. tuberculosis in different settings, and to identify Laboratory Procedures the clinical and social demographic factors associated with recent For suspected tuberculosis, 3 sputum samples collected at differ- transmission [1–5]. Patients with mycobacterial isolates that have ent time points (spot, early morning, and night) were examined identical genotypes were assigned to clusters and were assumed for acid-fast bacilli (AFB), and 2 of them were used for Lowen- to be caused by recent transmission, whereas those with a unique stein-Jensen culture. Sputum induction was used for patients who genotype represented reactivation of a remote infection [6]. had trouble producing a sputum sample spontaneously. China has a serious tuberculosis epidemic, with 1.4 million All of the M. tuberculosis isolates were sent to the provincial prevalent tuberculosis cases and 130 000 deaths among tuber- CDC for drug susceptibility testing to detect resistance to rifam- culosis cases annually [7]. Currently, the tuberculosis control pin (RIF) and isoniazid (INH) using the proportion method on program focuses on the proportion of patients with infectious Lowenstein-Jensen medium at the following concentrations: RIF tuberculosis disease who are cured by the end of their treat- 40 µg/mL and INH 0.2 µg/mL [11]. Multidrug-resistant (MDR) ment regimen. However, the prevalence of tuberculosis in a tuberculosis was defined as resistance to at least INH and RIF. population is also determined by the incidence of new tubercu- Bacterial genomic DNA was obtained from isolates by the losis cases and the duration of infectiousness [8]. As the duration boiled lysis method [12]. The Beijing genotype is the most prev- Downloaded from of infectiousness of undiagnosed, untreated individuals increases, alent family of M. tuberculosis strains in China [13].We used a set so does the likelihood that M. tuberculosis will transmit to others. of variable number tandem repeats (VNTR), which was opti- Although China has made significant achievements tackling the mized for both Beijing strains and other strains in China, had tuberculosis epidemic during the last few decades [9], the pre- high discriminatory power comparable to the IS6110–restriction fi valence of all pulmonary tuberculosis did not signi cantly de- fragment length polymorphism method [14],andincluded4 http://cid.oxfordjournals.org/ crease (from 466/100 000 in 2000 to 459/100 000 in 2010) [10]. hypervariable VNTR loci (VNTR3820, 1982, 3232, and 4120) Meanwhile, the relative contribution of recent transmission of that were in a consensus loci set for study recent transmission M. tuberculosis in China is unclear. [14, 15]. We used BioNumerics software version 5.0 (Applied We conducted a population-based molecular epidemiologic Maths, Belgium) to analyze the genotyping data. Tuberculosis study of pulmonary tuberculosis in 5 field sites in China to es- cases whose M. tuberculosis strains had an identical genotypic timate the magnitude of tuberculosis cases that were attributable pattern were considered a cluster, indicating recent transmis- to recent transmission of M. tuberculosis, to identify the risk fac- sion. Cases with a unique genotype pattern indicated reactiva- by guest on June 28, 2015 tors associated with recent transmission, and to estimate relative tion of latent tuberculosis [2]. We restricted the cluster analyses transmission rates. within the local study population in each respective site. Cross- contamination may have occurred if ≥2 isolates from different METHODS patients in the same region were processed on the same day in the laboratory and shared the same genotype. Study Population We performed a population-based molecular epidemiologic Data Collection study in 5 field sites in China from 1 July 2009 to 30 June At each field site, trained study workers recruited patients with 2012. In each of the 5 provinces, 1 county was selected as the tuberculosis; obtained their written informed consent; enrolled study site (Supplementary Figure 1). The 5 sites represent geo- them in the study; and conducted interviews using a standard- graphical areas and populations with different tuberculosis bur- ized questionnaire to collect information on demographic and dens and socioeconomic levels based on China’s census system clinical characteristics, medical history, and lifestyle behaviors. [10]. At each site, passive case finding was used to identify patients A diagnostic delay was defined as the time between a patient’s aged ≥15 years with suspected pulmonary tuberculosis with report of symptom onset and the date of confirmed diagnosis. symptoms, including cough for at least 2 weeks, fever, chest pain, Tuberculosis patients had a prior tuberculosis history