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Role of casual contacts in the recent transmission of tuberculosis in settings with high disease burden
ORIGINAL ARTICLE
BACTERIOLOGY
Role of casual contacts in the recent transmission of tuberculosis in settings with high disease burden W. Wang1,2, B. Mathema3,4, Y. Hu1,2, Q. Zhao1,2, W. Jiang1,2 and B. Xu1,2 1) Department of Epidemiology, School of Public Health, Fudan University, 2) Key Laboratory of Public Health Safety (Ministry of Education), Shanghai, China, 3) Department of Epidemiology, Mailman School of Public Health, Columbia University, New York, NY and 4) Tuberculosis Center, Public Health Research Institute, Newark, NJ, USA
Abstract Tuberculosis (TB) remains a major cause of morbidity and mortality worldwide. It is expected that combining multiple molecular methods will further help in focusing contact investigations. We performed a population-based molecular epidemiological study in six sites in China between 1 June 2009 and 31 December 2010. A genotyping method combining 7-loci MIRU-VNTR and IS6110-based RFLP was employed to determine predictors of recent transmission. A second interview was performed with the clustered patients to identify potential epidemiological links. The molecular clustering analysis revealed that 187 isolates (15.3%) were clustered by sharing identical VNTR-IS6110 combined patterns, with an estimated recent transmission index being 8.9%. None of these patients reported having contacts with other members within the same cluster. Nineteen of 121 reported having a history of contact with a TB case within 2 years before the current TB diagnosis. Additionally, geographical correlation was established for 19 cases in nine clusters, while only one possible epidemiological link was established in secondary interview. The results underscore the role of casual contact or reactivation of latent TB as a driving factor maintaining the current endemicity in rural China, with high disease burdens of tuberculosis. Keywords: Casual contacts, clustering, contact investigation, recent transmission, tuberculosis Original Submission: 19 April 2014; Revised Submission: 14 June 2014; Accepted: 14 June 2014
Editor: M. Paul Article published online: 19 June 2014 Clin Microbiol Infect 2014; 20: 1140–1145 10.1111/1469-0691.12726
Corresponding author: B. Xu, School of Public Health, Fudan University, 138 Yi Xue Yuan Road, Shanghai 200032, China E-mail: [email protected]
Tuberculosis (TB) remains a major cause of morbidity and mortality worldwide. Particularly worrisome is the worldwide emergence of multidrug-resistant (MDR) and extensively resistant forms of Mycobacterium tuberculosis (Mtb) strains that are difficult to treat and associated with poor therapeutic outcomes [1]. Globally in 2012, data from drug resistance surveys and continuous surveillance among notified TB cases suggest that 3.6% of newly diagnosed TB cases and 20% of those previously treated for TB had MDR-TB [2]. China has the second highest burden of TB worldwide, with c. 1.3 million new TB cases detected annually [3]. Additionally, China is one of the hot spots of MDR-TB, with a prevalence of MDR-TB of 5.7% and
25.6% among new and previously treated cases according to the latest national TB survey in 2008 [4]. Molecular tools have enhanced our understanding of TB epidemiology by providing insight into the transmission dynamics, source and spread of Mtb [5,6]. Moreover, molecular epidemiological methods have refined the estimates of recent transmission so that they are important indicators in assessing the effectiveness of TB control programmes [7,8] and identifying previously unrecognized epidemiological links [9,10]. Conversely, genotyping has highlighted some limitations of conventional contact investigations to identify recent transmission. For instance, a molecular epidemiological study suggested that interventions only for close contacts might be inadequate to identify recently infected patients if contact occurs outside the household or close relatives/friends [11]. In Rotterdam, molecular typing identified widespread transmission from multiple sources among drug users, illustrating the limitations of contact investigation in high-risk populations and
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prompting an active case-finding programme [12]. In various settings, a substantial proportion of household contacts were infected with a different strain to that of the index case: 30% in California [13] and 54% in Cape Town [14]. The utility of molecular methods in clarifying transmission patterns was heavily dependent on the half-life of biomarker(s) used [15]. As such, it is expected that combining multiple molecular methods such as restriction fragment length polymorphisms (RFLP) typing with biomarkers including mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR) will further help in focusing contact investigations. In the present work, we used MIRU-VNTR and IS6110-based RFLP genotyping to understand the transmission of TB in rural China by examining circulating M. tuberculosis strains and to determine the predictors of recent transmission.
Materials and Methods We performed a population-based molecular epidemiological study at six sites in China between 1 June 2009 and 31 December 2010 (Fig. 1). The field sites cover a total population of about 5.8 million inhabitants; 67% of them were part of a rural population, including three counties (Changshan [CS], Tengzhou [TZ] and Shenxian [SX]) in Shandong Province and three counties (Jianhu [JH], Guanyun [GY] and Ganyu [GYu]) in Jiangsu Province.
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Study population
Inclusion criteria were patients who had active TB that was bacteriologically confirmed by sputum culture and who provided informed consent for this study. Extra-pulmonary TB cases were excluded from the study. Ethical approval was given by the Ethics Committee of the School of Public Health, Fudan University. Data collection
Subjects were interviewed at the time of TB diagnosis at the county TB dispensaries (CTDs) by physicians who underwent a 2-day training course for the interview. A semi-structured questionnaire was developed that covered general demographic and socio-economic characteristics, clinical symptoms and disease history at TB diagnosis. BCG vaccination was determined by self-reporting and confirmed by the presence of a scar at interviewing. Family income was self-reported; the products that families produced during the same time were converted and added to the total income. A second interview was performed only with clustered patients to identify potential epidemiological links. Each of the clustered patients was interviewed again to obtain more-detailed data that were not recorded at the first interview (e.g. data regarding jobs, detailed migration/relocation information before the onset of TB, entertainment activities and related locations) and more information about known contacts with TB. Finally, the patients were asked whether they could recognize some or all of the patients clustered with them. During the interview, we asked the participants to give their oral consent to divulge their identity to other TB patients in their cluster. When patients were absent or refused to come, we telephoned the patients or their families to obtain their permission for the disclosure of their identity. Molecular characterization
FIG. 1. Number of cases with different epidemiological contact histories during the follow-ups.
The sputum samples of patients were sent to the reference laboratory in the Shandong Provincial TB Center for culture and drug susceptibility testing. Drug susceptibility testing was performed for isoniazid, rifampin, pyrazinamide and ethambutol on Lowenstein-Jensen solid medium. Drug-resistant TB refers to TB that is resistant to any of the four first-line anti-tuberculosis drugs. MDR-TB is defined as TB that is resistant to at least isoniazid and rifampicin. Mycobacterium tuberculosis genomic DNA was extracted in the laboratory by standard methods [16] and then shipped to Fudan University to genotype the M. tuberculosis isolates using a high-resolution 7-loci variable number of tandem repeats (7-VNTR) method that was described by Zhang et al. [17], showing sufficient discriminatory power to differentiate prevalent W-Beijing genotype strains in the Chinese popula-
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tion. Only one isolate per each patient was included in this study. Isolates with identical 7-VNTR genotypes were subjected to IS6110-based RFLP analysis. A combined cluster was defined as a series of isolates that had both the same VNTR allele profile and IS6110 fingerprint identity.
TABLE 1. Odds ratio and adjusted odds ratio (and 95% confidence interval) for risk factors for clustering (cluster/ unique) Clustered Total
Pulmonary cavities No 789 116 Yes 427 70 TB history Newly 1039 160 diagnosed Previously 183 27 treated Smear test Negative 248 34 Positive 974 153 Work away from hometown No or 970 134 missing Yes 252 53 Low-income householdc No 59 7 Yes 1163 180 Report contact with tuberculosis No 1213 183 Yes 8 1 MDR-TB No 1144 167 Yes 78 20 BCG No or 835 123 unknown Yes 387 64
Estimation of recent transmission
In order to estimate the extent of recent transmission of TB, c)/n, we calculated the widely used index RTIn 1 = (nc where n is the total number of cases in the sample, c is the number of genotypes represented by at least two cases, and nc is the total number of cases in a cluster of two or more [18]. Statistical analysis
The chi-squared test was used to assess differences in the proportion of M. tuberculosis isolates clustering with different genotypes; when the expected value was <5, Fisher’s exact two-tailed test was used. Variables were examined both in univariate analyses and the binary logistic model where the odds ratio (OR) for the proportion clustering was adjusted for age, sex and county. The family income was skewed, and the variable was therefore included in the logistic regression model after being separated into two groups by median. Statistical analysis was carried out by SPSS software 11.0 (SPSS Inc., Chicago, IL, USA).
No.
%
Crude OR (95% CI)a
Adjusted OR (95% CI)b
14.7 16.4
1 1.14 (0.80–1.53)
1 1.05 (0.75–1.50)
15.4
1
1
14.8
0.95 (0.61–1.48)
0.85 (0.51–1.41)
13.7 15.7
1 1.13 (0.76–1.67)
1 1.13 (0.75–1.71)
13.8
1
1
21.0
1.66 (1.17–2.37)‡
1.35 (0.91–2.01)
11.9 1 15.5 0.74 (0.53–1.03) patients at diagnosis 15.1 1 12.5 0.79 (0.10–6.46)
1 0.82 (0.57–1.16)
14.6 25.6
1 2.02 (1.18–3.44)†
1 2.16 (1.18–3.95)†
14.7
1
1
16.5
1.15 (0.83–1.60)
0.73 (0.47–1.15)
1 0.81 (0.09–7.00)
a
Estimated from univariate logistic model. b Estimated from multivariate logistic model, with adjustment for age and sex. c Low-income household referred to those that were living below median income. † p <0.05. ‡ p <0.01. MDR-TB, multidrug-resistant tuberculosis (resistant to at least isoniazid and rifampicin); BCG, Bacillus Calmette-Guerin.
Results Subject characteristics
From January 2007 through to December 2008, a total of 2221 registered pulmonary TB patients were recruited in the local TB dispensaries. After bacterial culture and strain identification, a total of 1546 VNTR patterns were successfully obtained. Subsequently, 1222 VNTR-IS6110 combined DNA fingerprints were obtained and included in the analysis (Table 1), including 275 (22.5%) in CS, 188 (15.4%) in TZ, 168 (13.7%) in SX, 150 (12.3%) in JH, 248 (20.3%) in GY and 193 (15.8%) in GYu. Another 40 isolates were non-tuberculosis Mycobacteria and two isolates were Mycobacterium bovis. The patients ranged from 16.2 to 88.9 years old, with a mean age of 48.4 years (Table 2). Clustering and recent transmission
The 7-VNTR-based genotyping indicated that 324 isolates (26.5%) shared identical VNTR-IS6110 patterns, which were referred to as the ‘cluster’ category; the remaining 898 isolates exhibited a unique pattern, and were referred to as unique category/isolates. In addition, the molecular clustering analysis combined 7-VNTR-based genotyping and IS6110-based RFLP
and revealed that 187 isolates (15.3%) were clustered. Of the 258 drug-resistant Mtb isolates, a total of 233 VNTR-IS6110 RFLP genotype patterns were observed, with 21 clusters composed of 46 drug-resistant isolates (16.7%). The total RTIn 1 was estimated at 8.9%. Of these clusters, 18 clusters were composed solely of 43 drug-resistant isolates, and the other three clusters were shared among three drug-resistant and three pan-susceptible isolates. The 20 MDR isolates (25.6%) represented 12 VNTR-IS6110 RFLP profiles, and the remaining 58 isolates exhibited unique genotypes, indicating a 10.3% RTIn 1 (vs. non-MDR isolates, p 0.01). Risk factors for recent transmission
To identify risk factors for recent infection with Mtb, patients harbouring clustered isolates were compared with patients whose isolates were not in clusters (Table 3). Univariate analysis indicated that TB patients who worked out of town (domestic migrant workers) during the 1 year prior to the investigation had a significantly higher rate of clustering than those who did not (21 vs.14%; OR, 1.66; 95% CI, 1.17–2.37). Patients infected with MDR Mtb were more likely to be clustered than those with non-MDR isolates. After adjustment
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TABLE 2. Contact information for the patients who had a history of contact with TB patients Case No
Cluster No
Epi-link
CA8058 CA8134 CA0102 CA0058 GA9035 GA8064
CA13 CA13 CA3 CA7 GA10 GA11
No No No No No No
GA0071
GA14
No
GA0055
GA19
No
GA0009
GA2
No
GU9099
GU11
No
GU0009
GU13
No
GU0003
GU14
No
JI8076 JI8080
JI2 JI5
Possible No
JI9127
JI5
No
JI9016
JI8
No
JI8028
JI9
No
XI8067
XI1
No
XI0032
XI7
No
Contact information Denied contact history with case in same cluster, but had contacts with other TB patients. Denied contact history with case in same cluster, but her husband was a TB patient. Denied contact history with case in same cluster, but had contacts with another TB patient. Denied contact history with case in same cluster, but had contacts with other TB patients. Denied contact history with case in same cluster, but had contacts with other TB patients. Denied contact history with case in same cluster, but had contacts with other TB patients (roommates) when working outside. Denied contact history with case in same cluster, but had contacts with another TB patient (a teacher). Denied contact history with case in same cluster, but had contacts with other TB patients (roommates), when working outside. Denied contact history with case in same cluster, but had contacts with other TB patients (neighbours). Denied contact history with case in same cluster, but had contacts with other TB patients (father and brothers). Denied contact history with case in same cluster, but had contacts with other TB patients when working as a computer repairman. Denied contact history with case in same cluster, but had contacts with other TB patients (father and roommates). Died of TB; his wife reported having history of contacts with the case in same cluster. Denied contact history with case in same cluster, but had contacts with another TB patient (a friend). Denied contact history with case in same cluster, but had contacts with another TB patient (a colleague). Denied contact history with case in same cluster, but had contacts with another TB patient (his father, who died of TB). Denied contact history with case in same cluster, but had contacts with other TB patients (neighbours). Denied contact history with case in same cluster, but had contacts with another TB patient (a friend, who he also frequently played card games with). Denied contact history with case in same cluster, but had contacts with other TB patients (a neighbour, who was detected as having TB 4 months prior to this case, and his brother).
Epi-link, epidemiological links; TB, tuberculosis.
TABLE 3. Estimation of recent transmission among the isolations Drug resistance Total Pan-sensitive Any drug (INH, IRF, STR or EMB) INH RIF STR EMB MDR-TB
No. of patients
Proportion (%)
No. of patients in the clustersa
No. of clustersa
Cluster sizea
Proportion clusteringa (%)
p value*
1222 964 258
100.0 78.9 21.1
187 141 46
78 55 21
1–4 1–4 1–3
15.3 14.6 16.7
0.143 <0.001
8.9 8.9 9.7
157 98 191 35 78
12.8 8.0 15.6 2.9 6.4
45 20 17 2 20
16 13 12 2 12
2–3 1–2 1–2 1–2 1–2
28.7 20.4 8.9 5.7 25.6
<0.001 0.143 0.004 0.077 0.010
18.5 7.1 2.6 0.0 10.3
RTIn
b 1
(%)
a
Determined by a combined genotyping of IS6110 RFLP and 7-VNTR. Estimated proportions of recent transmission, RTIn 1 = (nc c)/n, where n is the total number of cases in the sample, c is the number of genotypes represented by at least two cases, and nc is the total number of cases in cluster of size two or more [18]. *p value was obtained from the chi-square test. INH, isoniazid; IRF, rifampin, STR, streptomycin; EMB, ethambutol.
b
for age and sex, MDR remained significantly associated with clustering (OR, 2.16; 95% CI, 1.18–3.95). Epidemiological contacts and recent TB transmission
Of the 187 patient isolates in 70 clusters, 66 patients were lost to follow-up due to no response to the telephone call or death before the investigation. To determine epidemiological links, face-to-face interviews were conducted with the remaining 121 patients. While none of these 121 patients with successful follow-up reported having contacts with other members of the same cluster, 19 reported having a history of contact with a TB case who was not included in the current study cohort within 2 years before the current TB diagnosis. Additionally, geo-
graphical correlation was established for 19 cases in nine clusters. Only one possible epidemiological link was established in a deceased patient whose wife reported that the patient had worked in the same factory as other patients within the cluster (Table 2).
Discussion We used a combination of molecular methods and epidemiological data to determine the genetic diversity of circulating Mtb strains, and provide the estimates of recent transmission and risk factors for clustering among pulmonary TB patients in rural
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China, where on average 67% of the population belonged to an agricultural population. A recent nationwide survey showed that the prevalence of TB was relatively higher in rural areas than in urban areas, which is not the case in low-incidence countries [19]. The surprisingly low clustering found in this study, in contrast to places such as South Africa, which has co-infection of TB and human immunodeficiency virus (HIV) and explosive outbreaks (i.e. large cluster sizes), suggests that casual contact may be more important than previously thought. In China, case finding for TB is mainly passive. Although being a household contact of a TB patient is a strong risk factor [20,21], in high-incidence settings most cases of TB are not attributable to household contact [22]. Therefore the importance of casual contact in TB transmission is not surprising because many people exposed to a small risk can account for more cases than a few exposed to a large risk [23]. The total rate of clustering (15%), which is substantially lower than estimates reported in other studies conducted in low-incidence areas (e.g. Denmark (49%) [24], New York (48%) [25], Spain (36%) [26], Germany (49%) [27], San Francisco (70%) [13] and elsewhere in the United States (71%) [28]), is also lower than most of the other reported figures in China, ranging from 10% to 32% [29–31]. Using the n 1 method, 9% of cases were likely to be due to recent transmission in this region. Although inconsistent, studies increasingly report that MDR strains are one of the risk factors for recent transmission [32], a notion supported by our findings. Traditional approaches have shown that certain groups are at higher risk for TB (e.g. elderly people, prisoners, the homeless and injecting drug users) and non-traditional settings where these groups congregate are important in facilitating TB transmission [33]. In contrast to those findings, contacts within and outside the family and household were less likely to be confirmed as sources of infection [28]. Moreover, no place or occasion could be confirmed as the source or geographical point of transmission, even in sites that had a relatively enclosed population. Mtb genotyping techniques offer a method for TB prevention and control [34] by demonstrating previously unrecognized linkages even within endemic communities. The ability of Mtb genotyping to estimate recent TB transmission, however, has been both asserted and challenged [10]. In the current study, we found that 27% of the isolates shared 7-VNTR fingerprint patterns; when the IS6110 was combined, however, it became 15%. The combination of 7-VNTR and RFLP increased the Hunter-Gaston discriminatory index and therefore decreased the proportion of cases attributed to recent transmission. A previous study indicated that the HGI value of 7-VNTR was slightly lower than that of IS6110 RFLP genotyping in China, thus requiring secondary genotyping of clustered strains (e.g. IS6110 RFLP) to efficiently differentiate Beijing genotype strains [17].
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This study identified small cluster sizes ranging from two to four patients, indicating limited scale of transmission in this high-incidence rural area. Low clustering rates combined with small cluster sizes may indicate a more vital role of reactive disease compared with recent transmission. The disparities in the scale of clustering between countries with a high and low TB disease burden appear obvious [35]. However, the mechanisms to explain the difference are complex. Unlike in other high-incidence countries, where TB patients in urban areas were more likely to be clustered [36], those patients in a rural setting in China were subjected to a higher risk of clustering. This disparity may be partly driven by the frequent population movement in China’s urban areas [37], and therefore it is usually difficult to obtain an entire profile of TB transmission. Patients for this study were those who registered in the CTDs; this sample represented about 80% of the patients in the studied communities according to the estimates from a recent household screening study during the same period [38]. Therefore, some potential source cases may have been missed in those communities, thereby underestimating the actual extent of recent transmission. Lastly, although the 7-VNTR genotyping method was reported to be less discriminating relative to IS6110 RFLP for isolates predominating in China, the combined genotyping method employed in this study may have still underestimated the rate of clustering.
Conclusion In conclusion, in rural China, where remote reactivated TB is the main concern as indicated by the low rate of clustering, few epidemiological links could be established among those in molecular clusters, underscoring the role of casual contacts or reactivation of latent TB as a driving factor maintaining the current endemicity in China. This suggests the need for prevention strategies (e.g. isoniazid preventive therapy) to reduce active TB in people with latent TB infection, which has also been proposed by the World Health Organization [39].
Acknowledgements This study received a grant from the US National Institutes of Health/National Institute of Allergy and Infectious Diseases (No. 1R01AI075463).
Transparency Declaration The authors declare that they have no competing interests.
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BACTERIOLOGY
Role of casual contacts in the recent transmission of tuberculosis in settings with high disease burden W. Wang1,2, B. Mathema3,4, Y. Hu1,2, Q. Zhao1,2, W. Jiang1,2 and B. Xu1,2 1) Department of Epidemiology, School of Public Health, Fudan University, 2) Key Laboratory of Public Health Safety (Ministry of Education), Shanghai, China, 3) Department of Epidemiology, Mailman School of Public Health, Columbia University, New York, NY and 4) Tuberculosis Center, Public Health Research Institute, Newark, NJ, USA
Abstract Tuberculosis (TB) remains a major cause of morbidity and mortality worldwide. It is expected that combining multiple molecular methods will further help in focusing contact investigations. We performed a population-based molecular epidemiological study in six sites in China between 1 June 2009 and 31 December 2010. A genotyping method combining 7-loci MIRU-VNTR and IS6110-based RFLP was employed to determine predictors of recent transmission. A second interview was performed with the clustered patients to identify potential epidemiological links. The molecular clustering analysis revealed that 187 isolates (15.3%) were clustered by sharing identical VNTR-IS6110 combined patterns, with an estimated recent transmission index being 8.9%. None of these patients reported having contacts with other members within the same cluster. Nineteen of 121 reported having a history of contact with a TB case within 2 years before the current TB diagnosis. Additionally, geographical correlation was established for 19 cases in nine clusters, while only one possible epidemiological link was established in secondary interview. The results underscore the role of casual contact or reactivation of latent TB as a driving factor maintaining the current endemicity in rural China, with high disease burdens of tuberculosis. Keywords: Casual contacts, clustering, contact investigation, recent transmission, tuberculosis Original Submission: 19 April 2014; Revised Submission: 14 June 2014; Accepted: 14 June 2014
Editor: M. Paul Article published online: 19 June 2014 Clin Microbiol Infect 2014; 20: 1140–1145 10.1111/1469-0691.12726
Corresponding author: B. Xu, School of Public Health, Fudan University, 138 Yi Xue Yuan Road, Shanghai 200032, China E-mail: [email protected]
Tuberculosis (TB) remains a major cause of morbidity and mortality worldwide. Particularly worrisome is the worldwide emergence of multidrug-resistant (MDR) and extensively resistant forms of Mycobacterium tuberculosis (Mtb) strains that are difficult to treat and associated with poor therapeutic outcomes [1]. Globally in 2012, data from drug resistance surveys and continuous surveillance among notified TB cases suggest that 3.6% of newly diagnosed TB cases and 20% of those previously treated for TB had MDR-TB [2]. China has the second highest burden of TB worldwide, with c. 1.3 million new TB cases detected annually [3]. Additionally, China is one of the hot spots of MDR-TB, with a prevalence of MDR-TB of 5.7% and
25.6% among new and previously treated cases according to the latest national TB survey in 2008 [4]. Molecular tools have enhanced our understanding of TB epidemiology by providing insight into the transmission dynamics, source and spread of Mtb [5,6]. Moreover, molecular epidemiological methods have refined the estimates of recent transmission so that they are important indicators in assessing the effectiveness of TB control programmes [7,8] and identifying previously unrecognized epidemiological links [9,10]. Conversely, genotyping has highlighted some limitations of conventional contact investigations to identify recent transmission. For instance, a molecular epidemiological study suggested that interventions only for close contacts might be inadequate to identify recently infected patients if contact occurs outside the household or close relatives/friends [11]. In Rotterdam, molecular typing identified widespread transmission from multiple sources among drug users, illustrating the limitations of contact investigation in high-risk populations and
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prompting an active case-finding programme [12]. In various settings, a substantial proportion of household contacts were infected with a different strain to that of the index case: 30% in California [13] and 54% in Cape Town [14]. The utility of molecular methods in clarifying transmission patterns was heavily dependent on the half-life of biomarker(s) used [15]. As such, it is expected that combining multiple molecular methods such as restriction fragment length polymorphisms (RFLP) typing with biomarkers including mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR) will further help in focusing contact investigations. In the present work, we used MIRU-VNTR and IS6110-based RFLP genotyping to understand the transmission of TB in rural China by examining circulating M. tuberculosis strains and to determine the predictors of recent transmission.
Materials and Methods We performed a population-based molecular epidemiological study at six sites in China between 1 June 2009 and 31 December 2010 (Fig. 1). The field sites cover a total population of about 5.8 million inhabitants; 67% of them were part of a rural population, including three counties (Changshan [CS], Tengzhou [TZ] and Shenxian [SX]) in Shandong Province and three counties (Jianhu [JH], Guanyun [GY] and Ganyu [GYu]) in Jiangsu Province.
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Study population
Inclusion criteria were patients who had active TB that was bacteriologically confirmed by sputum culture and who provided informed consent for this study. Extra-pulmonary TB cases were excluded from the study. Ethical approval was given by the Ethics Committee of the School of Public Health, Fudan University. Data collection
Subjects were interviewed at the time of TB diagnosis at the county TB dispensaries (CTDs) by physicians who underwent a 2-day training course for the interview. A semi-structured questionnaire was developed that covered general demographic and socio-economic characteristics, clinical symptoms and disease history at TB diagnosis. BCG vaccination was determined by self-reporting and confirmed by the presence of a scar at interviewing. Family income was self-reported; the products that families produced during the same time were converted and added to the total income. A second interview was performed only with clustered patients to identify potential epidemiological links. Each of the clustered patients was interviewed again to obtain more-detailed data that were not recorded at the first interview (e.g. data regarding jobs, detailed migration/relocation information before the onset of TB, entertainment activities and related locations) and more information about known contacts with TB. Finally, the patients were asked whether they could recognize some or all of the patients clustered with them. During the interview, we asked the participants to give their oral consent to divulge their identity to other TB patients in their cluster. When patients were absent or refused to come, we telephoned the patients or their families to obtain their permission for the disclosure of their identity. Molecular characterization
FIG. 1. Number of cases with different epidemiological contact histories during the follow-ups.
The sputum samples of patients were sent to the reference laboratory in the Shandong Provincial TB Center for culture and drug susceptibility testing. Drug susceptibility testing was performed for isoniazid, rifampin, pyrazinamide and ethambutol on Lowenstein-Jensen solid medium. Drug-resistant TB refers to TB that is resistant to any of the four first-line anti-tuberculosis drugs. MDR-TB is defined as TB that is resistant to at least isoniazid and rifampicin. Mycobacterium tuberculosis genomic DNA was extracted in the laboratory by standard methods [16] and then shipped to Fudan University to genotype the M. tuberculosis isolates using a high-resolution 7-loci variable number of tandem repeats (7-VNTR) method that was described by Zhang et al. [17], showing sufficient discriminatory power to differentiate prevalent W-Beijing genotype strains in the Chinese popula-
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tion. Only one isolate per each patient was included in this study. Isolates with identical 7-VNTR genotypes were subjected to IS6110-based RFLP analysis. A combined cluster was defined as a series of isolates that had both the same VNTR allele profile and IS6110 fingerprint identity.
TABLE 1. Odds ratio and adjusted odds ratio (and 95% confidence interval) for risk factors for clustering (cluster/ unique) Clustered Total
Pulmonary cavities No 789 116 Yes 427 70 TB history Newly 1039 160 diagnosed Previously 183 27 treated Smear test Negative 248 34 Positive 974 153 Work away from hometown No or 970 134 missing Yes 252 53 Low-income householdc No 59 7 Yes 1163 180 Report contact with tuberculosis No 1213 183 Yes 8 1 MDR-TB No 1144 167 Yes 78 20 BCG No or 835 123 unknown Yes 387 64
Estimation of recent transmission
In order to estimate the extent of recent transmission of TB, c)/n, we calculated the widely used index RTIn 1 = (nc where n is the total number of cases in the sample, c is the number of genotypes represented by at least two cases, and nc is the total number of cases in a cluster of two or more [18]. Statistical analysis
The chi-squared test was used to assess differences in the proportion of M. tuberculosis isolates clustering with different genotypes; when the expected value was <5, Fisher’s exact two-tailed test was used. Variables were examined both in univariate analyses and the binary logistic model where the odds ratio (OR) for the proportion clustering was adjusted for age, sex and county. The family income was skewed, and the variable was therefore included in the logistic regression model after being separated into two groups by median. Statistical analysis was carried out by SPSS software 11.0 (SPSS Inc., Chicago, IL, USA).
No.
%
Crude OR (95% CI)a
Adjusted OR (95% CI)b
14.7 16.4
1 1.14 (0.80–1.53)
1 1.05 (0.75–1.50)
15.4
1
1
14.8
0.95 (0.61–1.48)
0.85 (0.51–1.41)
13.7 15.7
1 1.13 (0.76–1.67)
1 1.13 (0.75–1.71)
13.8
1
1
21.0
1.66 (1.17–2.37)‡
1.35 (0.91–2.01)
11.9 1 15.5 0.74 (0.53–1.03) patients at diagnosis 15.1 1 12.5 0.79 (0.10–6.46)
1 0.82 (0.57–1.16)
14.6 25.6
1 2.02 (1.18–3.44)†
1 2.16 (1.18–3.95)†
14.7
1
1
16.5
1.15 (0.83–1.60)
0.73 (0.47–1.15)
1 0.81 (0.09–7.00)
a
Estimated from univariate logistic model. b Estimated from multivariate logistic model, with adjustment for age and sex. c Low-income household referred to those that were living below median income. † p <0.05. ‡ p <0.01. MDR-TB, multidrug-resistant tuberculosis (resistant to at least isoniazid and rifampicin); BCG, Bacillus Calmette-Guerin.
Results Subject characteristics
From January 2007 through to December 2008, a total of 2221 registered pulmonary TB patients were recruited in the local TB dispensaries. After bacterial culture and strain identification, a total of 1546 VNTR patterns were successfully obtained. Subsequently, 1222 VNTR-IS6110 combined DNA fingerprints were obtained and included in the analysis (Table 1), including 275 (22.5%) in CS, 188 (15.4%) in TZ, 168 (13.7%) in SX, 150 (12.3%) in JH, 248 (20.3%) in GY and 193 (15.8%) in GYu. Another 40 isolates were non-tuberculosis Mycobacteria and two isolates were Mycobacterium bovis. The patients ranged from 16.2 to 88.9 years old, with a mean age of 48.4 years (Table 2). Clustering and recent transmission
The 7-VNTR-based genotyping indicated that 324 isolates (26.5%) shared identical VNTR-IS6110 patterns, which were referred to as the ‘cluster’ category; the remaining 898 isolates exhibited a unique pattern, and were referred to as unique category/isolates. In addition, the molecular clustering analysis combined 7-VNTR-based genotyping and IS6110-based RFLP
and revealed that 187 isolates (15.3%) were clustered. Of the 258 drug-resistant Mtb isolates, a total of 233 VNTR-IS6110 RFLP genotype patterns were observed, with 21 clusters composed of 46 drug-resistant isolates (16.7%). The total RTIn 1 was estimated at 8.9%. Of these clusters, 18 clusters were composed solely of 43 drug-resistant isolates, and the other three clusters were shared among three drug-resistant and three pan-susceptible isolates. The 20 MDR isolates (25.6%) represented 12 VNTR-IS6110 RFLP profiles, and the remaining 58 isolates exhibited unique genotypes, indicating a 10.3% RTIn 1 (vs. non-MDR isolates, p 0.01). Risk factors for recent transmission
To identify risk factors for recent infection with Mtb, patients harbouring clustered isolates were compared with patients whose isolates were not in clusters (Table 3). Univariate analysis indicated that TB patients who worked out of town (domestic migrant workers) during the 1 year prior to the investigation had a significantly higher rate of clustering than those who did not (21 vs.14%; OR, 1.66; 95% CI, 1.17–2.37). Patients infected with MDR Mtb were more likely to be clustered than those with non-MDR isolates. After adjustment
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TABLE 2. Contact information for the patients who had a history of contact with TB patients Case No
Cluster No
Epi-link
CA8058 CA8134 CA0102 CA0058 GA9035 GA8064
CA13 CA13 CA3 CA7 GA10 GA11
No No No No No No
GA0071
GA14
No
GA0055
GA19
No
GA0009
GA2
No
GU9099
GU11
No
GU0009
GU13
No
GU0003
GU14
No
JI8076 JI8080
JI2 JI5
Possible No
JI9127
JI5
No
JI9016
JI8
No
JI8028
JI9
No
XI8067
XI1
No
XI0032
XI7
No
Contact information Denied contact history with case in same cluster, but had contacts with other TB patients. Denied contact history with case in same cluster, but her husband was a TB patient. Denied contact history with case in same cluster, but had contacts with another TB patient. Denied contact history with case in same cluster, but had contacts with other TB patients. Denied contact history with case in same cluster, but had contacts with other TB patients. Denied contact history with case in same cluster, but had contacts with other TB patients (roommates) when working outside. Denied contact history with case in same cluster, but had contacts with another TB patient (a teacher). Denied contact history with case in same cluster, but had contacts with other TB patients (roommates), when working outside. Denied contact history with case in same cluster, but had contacts with other TB patients (neighbours). Denied contact history with case in same cluster, but had contacts with other TB patients (father and brothers). Denied contact history with case in same cluster, but had contacts with other TB patients when working as a computer repairman. Denied contact history with case in same cluster, but had contacts with other TB patients (father and roommates). Died of TB; his wife reported having history of contacts with the case in same cluster. Denied contact history with case in same cluster, but had contacts with another TB patient (a friend). Denied contact history with case in same cluster, but had contacts with another TB patient (a colleague). Denied contact history with case in same cluster, but had contacts with another TB patient (his father, who died of TB). Denied contact history with case in same cluster, but had contacts with other TB patients (neighbours). Denied contact history with case in same cluster, but had contacts with another TB patient (a friend, who he also frequently played card games with). Denied contact history with case in same cluster, but had contacts with other TB patients (a neighbour, who was detected as having TB 4 months prior to this case, and his brother).
Epi-link, epidemiological links; TB, tuberculosis.
TABLE 3. Estimation of recent transmission among the isolations Drug resistance Total Pan-sensitive Any drug (INH, IRF, STR or EMB) INH RIF STR EMB MDR-TB
No. of patients
Proportion (%)
No. of patients in the clustersa
No. of clustersa
Cluster sizea
Proportion clusteringa (%)
p value*
1222 964 258
100.0 78.9 21.1
187 141 46
78 55 21
1–4 1–4 1–3
15.3 14.6 16.7
0.143 <0.001
8.9 8.9 9.7
157 98 191 35 78
12.8 8.0 15.6 2.9 6.4
45 20 17 2 20
16 13 12 2 12
2–3 1–2 1–2 1–2 1–2
28.7 20.4 8.9 5.7 25.6
<0.001 0.143 0.004 0.077 0.010
18.5 7.1 2.6 0.0 10.3
RTIn
b 1
(%)
a
Determined by a combined genotyping of IS6110 RFLP and 7-VNTR. Estimated proportions of recent transmission, RTIn 1 = (nc c)/n, where n is the total number of cases in the sample, c is the number of genotypes represented by at least two cases, and nc is the total number of cases in cluster of size two or more [18]. *p value was obtained from the chi-square test. INH, isoniazid; IRF, rifampin, STR, streptomycin; EMB, ethambutol.
b
for age and sex, MDR remained significantly associated with clustering (OR, 2.16; 95% CI, 1.18–3.95). Epidemiological contacts and recent TB transmission
Of the 187 patient isolates in 70 clusters, 66 patients were lost to follow-up due to no response to the telephone call or death before the investigation. To determine epidemiological links, face-to-face interviews were conducted with the remaining 121 patients. While none of these 121 patients with successful follow-up reported having contacts with other members of the same cluster, 19 reported having a history of contact with a TB case who was not included in the current study cohort within 2 years before the current TB diagnosis. Additionally, geo-
graphical correlation was established for 19 cases in nine clusters. Only one possible epidemiological link was established in a deceased patient whose wife reported that the patient had worked in the same factory as other patients within the cluster (Table 2).
Discussion We used a combination of molecular methods and epidemiological data to determine the genetic diversity of circulating Mtb strains, and provide the estimates of recent transmission and risk factors for clustering among pulmonary TB patients in rural
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China, where on average 67% of the population belonged to an agricultural population. A recent nationwide survey showed that the prevalence of TB was relatively higher in rural areas than in urban areas, which is not the case in low-incidence countries [19]. The surprisingly low clustering found in this study, in contrast to places such as South Africa, which has co-infection of TB and human immunodeficiency virus (HIV) and explosive outbreaks (i.e. large cluster sizes), suggests that casual contact may be more important than previously thought. In China, case finding for TB is mainly passive. Although being a household contact of a TB patient is a strong risk factor [20,21], in high-incidence settings most cases of TB are not attributable to household contact [22]. Therefore the importance of casual contact in TB transmission is not surprising because many people exposed to a small risk can account for more cases than a few exposed to a large risk [23]. The total rate of clustering (15%), which is substantially lower than estimates reported in other studies conducted in low-incidence areas (e.g. Denmark (49%) [24], New York (48%) [25], Spain (36%) [26], Germany (49%) [27], San Francisco (70%) [13] and elsewhere in the United States (71%) [28]), is also lower than most of the other reported figures in China, ranging from 10% to 32% [29–31]. Using the n 1 method, 9% of cases were likely to be due to recent transmission in this region. Although inconsistent, studies increasingly report that MDR strains are one of the risk factors for recent transmission [32], a notion supported by our findings. Traditional approaches have shown that certain groups are at higher risk for TB (e.g. elderly people, prisoners, the homeless and injecting drug users) and non-traditional settings where these groups congregate are important in facilitating TB transmission [33]. In contrast to those findings, contacts within and outside the family and household were less likely to be confirmed as sources of infection [28]. Moreover, no place or occasion could be confirmed as the source or geographical point of transmission, even in sites that had a relatively enclosed population. Mtb genotyping techniques offer a method for TB prevention and control [34] by demonstrating previously unrecognized linkages even within endemic communities. The ability of Mtb genotyping to estimate recent TB transmission, however, has been both asserted and challenged [10]. In the current study, we found that 27% of the isolates shared 7-VNTR fingerprint patterns; when the IS6110 was combined, however, it became 15%. The combination of 7-VNTR and RFLP increased the Hunter-Gaston discriminatory index and therefore decreased the proportion of cases attributed to recent transmission. A previous study indicated that the HGI value of 7-VNTR was slightly lower than that of IS6110 RFLP genotyping in China, thus requiring secondary genotyping of clustered strains (e.g. IS6110 RFLP) to efficiently differentiate Beijing genotype strains [17].
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This study identified small cluster sizes ranging from two to four patients, indicating limited scale of transmission in this high-incidence rural area. Low clustering rates combined with small cluster sizes may indicate a more vital role of reactive disease compared with recent transmission. The disparities in the scale of clustering between countries with a high and low TB disease burden appear obvious [35]. However, the mechanisms to explain the difference are complex. Unlike in other high-incidence countries, where TB patients in urban areas were more likely to be clustered [36], those patients in a rural setting in China were subjected to a higher risk of clustering. This disparity may be partly driven by the frequent population movement in China’s urban areas [37], and therefore it is usually difficult to obtain an entire profile of TB transmission. Patients for this study were those who registered in the CTDs; this sample represented about 80% of the patients in the studied communities according to the estimates from a recent household screening study during the same period [38]. Therefore, some potential source cases may have been missed in those communities, thereby underestimating the actual extent of recent transmission. Lastly, although the 7-VNTR genotyping method was reported to be less discriminating relative to IS6110 RFLP for isolates predominating in China, the combined genotyping method employed in this study may have still underestimated the rate of clustering.
Conclusion In conclusion, in rural China, where remote reactivated TB is the main concern as indicated by the low rate of clustering, few epidemiological links could be established among those in molecular clusters, underscoring the role of casual contacts or reactivation of latent TB as a driving factor maintaining the current endemicity in China. This suggests the need for prevention strategies (e.g. isoniazid preventive therapy) to reduce active TB in people with latent TB infection, which has also been proposed by the World Health Organization [39].
Acknowledgements This study received a grant from the US National Institutes of Health/National Institute of Allergy and Infectious Diseases (No. 1R01AI075463).
Transparency Declaration The authors declare that they have no competing interests.
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