Spatial Clustering Of Tuberculosis Patients And Genetic Diversity Of Mycobacterium Tuberculosis In Eastern China

Tuberculosis (TB) is an infectious diseases spread through the air caused by Mycobacterium tuberculosis (Mtb). Since 1990, along with frequent population flows, increased epidemics of drug-resistant TB and growth of immunodeficiency diseases such as AIDS, TB has become an important issue in public health after several decades of silence with the increasing prevalence around the world. China is one of 22 countries with the highest TB burden, having the second largest total TB cases and being one of the world’s 27 highest TB drug resistant burden countries.Most researches on TB are limited in single description of disease incidence, while they ignored the geographic correlation of tuberculosis incidence. TB, as an common infectious disease in China, is potentially associated with local environment, population, climate changes, etc.. Spatial epidemiology has important value to study the spatial distribution and influence factors of TB, and to develop TB prevention and control strategies. Due to the difficulty to detect latent infection and interaction of human and Mtb in TB epidemics, the traditional methods of epidemiological study cannot meet the needs to trace the TB infection sources and clarify the route of transmission. However, the molecular epidemiology has advantages to tackle the above epidemiological questions. The combination of spatial epidemiology and molecular epidemiology provides a new idea for studying TB. This research used spatial techniques and molecular epidemiology to explore spatial distribution of TB patients and molecular characterization of Mtb in rural areas of eastern China in order to better understand the TB transmission and provide evidence for policy making for effective TB prevention and control strategies.Methodology and main results:1. The spatial distribution and aggregation of TBDuring the study period, there were 680 and 445 active pulmonary TB patients reported from Funing and Yinzhou respectively. Geographic information system (GIS) was used for mapping and spatial autocorrelation analysis. SaTScan (version 8) was used to conduct the spatial scan statistic analysis. The global Moran’s I value of Funing county was 0.046648 (.P=0.593). The global Moran’s I value of Yinzhou District was 0.41023 (P=0.039). Local Moran’ s I values indicated there were local spatial autocorrelations in Yangji Town and Shiqi Town. Yangji Town and its peripheral regions were TB low incidence areas, while Shiqi Town and its peripheral regions had a TB high incidence. In Funing county, purely spatial scan statistic analysis showed that Guoshu Town was the only town where a geographical cluster was identified (RR=1.89,P=0.036), while Yizhou District had three geographical clusters (Xiaying, Shiqi and Wuxiang Town).2. The genetic diversity and clustering characteristics of MtbDuring the study peiod, 199 and 179 Mtb stains were collected from Funing and Yinzhou, respectively. The 378 Mtb strains were genotyped by 15 loci VNTR-MIRU. Genetic diversity of alleles varied between 0.0807 (ETR C) and 0.8056 (MIRU 26). The total Hunter-Gaston discriminatory index (HGDI) of 15 loci VNTR-MIRU for all strains was 0.9997. The HGDIs of 15 loci were sorted as:MIRU26> Qubllb> Mtub21> MIRU31> Qub26> Mtub04> MIRU10 MIRU40> Mtub30> Mtub39> Qub4156> ETR A> MIRU16> MIRU4> ETR C. In total, 359 strains out of 378 strains showed unique genotype while 32 strains shared 14 clusters. As a result, the proportion of clustering was 8.47%; the proportion of cases due to recent transmission was estimated at 4.76%. Resident TB patients were more likely to be clusterd than the non-resident TB patients (x2=11.458, P=0.001).3. VNTR-MIRU and SNP analysis in the temporal and spatial evolution of Beijing strains.Among 199 strains in Funing,182 strains (91.5%) were identified as Beijing Mtb strains using RD105 DTM-PCR typing. Beijing Mtb family strains were further classified into subtypes by SNP typing. At the same time, published information on the 15 loci VNTR-MIRU, IS6110-RFLP and SNP of Beijing family strains from Chongming (Shanghai), Taiwan and Japan were collected. We found that loci Mtub21, MIRU31, Qubllb, Mtub39, Mtub30 and MIRU40 were highly correlated with the subtypes of Beijing family, indicating their ability to work as molecular markers to identify the Beijing family. ST10 accounted for 53.4% and 53.3% of Beijing family strains in Funing and Taiwan respectively, while the largest subtype in Japan was ST19. The structure of MST and subtypes defined by SNP typing were consistent overall but different in part. Comparing the genotype of Funing with that of Chongming, Taiwan and Japan, we found that the genetic distance of Funing strains and Chongming strains was relatively close, while it was far between Funing strains, Taiwan strains and Japan strains. However, the genetic distance of modern genotypes of Chongming strains, Taiwan strains and Japan strains were close.4. The transmission mechanism of TB in rural areas of eastern ChinaWith combining spatial information and genotype, we used the ArcGIS 9.0 to map and analyze the spatial distribution characteristics of TB. TB patients with same genotype did not show close spatial location. Modern Bejing genotype had great popular advantage and was dominant in most of the towns except Chenliang Town, Shuoji Town, Lupu Town, Banhu Town and Luoqiao Town. The ST10 dominated (>50%) in areas with recent transmission, while it was less detected (<50%) in areas without recent transmission except Lupu town.Conclusions:1. The incidence of TB in rural areas of eastern China is subject to a positive local spatial autocorrelation. Yangji Town in Funing County and Xingying, Shiqi, Wuxiang Town in Yinzhou District were geographical clusters of high TB incidence,.2. An increased genetic diversity of VNTR-MIRU loci was observed. The discriminatory power of VNTR-MIRU could be adjusted by different loci combination. The longer phylogenetic distance between strains was, the higher discriminatory power the VNTR-MIRU genotype had.3. Loci Mtub21, MIRU31, Qubllb, Mtub39, Mtub30 and MIRU40 can be used as a reliable molecular marker in Mtb phylogenetic analysis. The evolution of Beijing family was closely interacted with human migration and society development. Along with the globalization, modern genotype was easier to expand in eastern Asia even the world.4. Sporadic transmission of TB might have played an important role in the transmission of TB in rural areas of eastern China. Recent transmission did not explain most geospatial clustering, suggesting that geospatial clustering largely depend on environment factors and social determinants. In Beijing strains, modern genotype especially ST10 had great popular advantage, suggesting that this genotype may have strong pathogenicity and was susceptible to infection and transmission.