| Mycobacterium tuberculosis (M. tuberculosis) is a pathogenic bacterium of tuberculosis. During the acid-fast staining, M. tuberculosis resists the acidic alcohol de-staining. In1882, German bacteriologist Robert Koch first confirmed that M tuberculosis was the pathogen of tuberculosis and that this pathogen was capable of infecting all the organs/tissues within human body and the lung was the most commonly infected organ. According to WHO’s estimation, there are approximately8-10millions of newly occurring tuberculosis world-wide and at least2-3million patients die of tuberculosis each year. Due to AIDS, narcotics-taking, drunk-drinking, poverty, use of immunosuppressors, dynamic population and occurrence of new drug-resistant strains of M. tuberculosis, there has been an increasing trend of tuberculosis incidence during the late20th century and the early21th century. This trend is continuously increased due to the limited availability of anti-tuberculosis drugs. The drug-resistance of M. tuberculosis has become an increasingly serious problem. The occurrence of multiple drug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (EXDR-TB) has brought new challenges in control of tuberculosis. The epidemic situation in China has been more serious and China is one of22countries that have high burdens of tuberculosis and is one of27countries that have high burdens of multiple-drug resistant/extensively drug-resistant tuberculosis (MDR/XDR-TB).The pathogenesis of tuberculosis is the consequences of cooperative actions of many factors. Currently, while one third of the total global population has been infected by M. tuberculosis, only one tenth of them get tuberculosis. It has been thought from previous studies that the courses of the pathogenesis of tuberculosis were mainly due to bacterial factors and environmental/social factors. However, more and more studies have demonstrated that the host factor is also an important for the pathogenesis of tuberculosis. Currently, it is believed that the pathogenesis of tuberculosis is the consequence of the comprehensive actions of host, pathogen, and environmental factors. A number of studies have indicated that genetic factors susceptible to tuberculosis play crucial roles in the pathogenesis of tuberculosis and these factors affect the resistance or susceptibility to tuberculosis and thus, cause the individual differences in the susceptibility to tuberculosis. Khor et al. found that the single nucleotide polymorphism (SNPs) of the gene encoding Cytokine-Inducible SRC homology2(SH2) domain protein (CISH) was related to the susceptibility of a number of diseases including tuberculosis. The polymorphic locus and polymorphic frequency of this gene are different among different diseases and different human populations. CISH is a member of the suppressor of cytokine signaling (SOCS) family, which regulates the signal pathways of a number of cytokines via its competition with STAT5for the binding site of the cytokine receptors and mainly influences IL-2signal transduction. Considering the key role of IL-2in immuno-resistance to tuberculosis, it is reasonable to speculate that CISH may play a crucial role in the pathogenesis of tuberculosis.In the present study, we performed in vitro drug sensitivity test of M. tuberculosis strains collected from1432cases of inpatients of Shanghai Pulmonary Hospital to investigate the epidemic situation of the drug resistant tuberculosis in Shanghai area. We also performed case-control study and collected the data of511cases of first time-treated patients with confirmed tuberculosis and500cases of health control and investigated the relationships between the population’s nutritional parameters, social and environmental factors with pathogenesis of tuberculosis. We performed the SNPs typing of CISH gene with the blood samples collected from898cases of patients with confirmed tuberculosis and825cases of health participants as the control by using the SNaPshot technology and investigated the relationship of polymorphisms of CISH gene among Chinese Han nationality and the susceptibility of tuberculosis in Shanghai area, and investigated the role of polymorphisms of CISH gene in the pathogenesis of tuberculosis.Part I:Study on the current situation of drug-resistant M. tuberculosis in Shanghai areaBeing one of the countries that have high burden of tuberculosis, the incidence of multiple-drug resistant/extensively drug-resistant tuberculosis (MDR/XDR-TB) in China has been continually increased. We performed the drug-resistant analysis of blood samples collected from1432patients who had confirmed tuberculosis during January2009to December2010by Shanghai Pulmonary Hospital and compared rate of drug-resistance of the first-time treated patients with that of the repeatedly treated patients.In this study, we collected and performed culture isolation of strains of M. tuberculosis from1432patients who were admitted and treated by Shanghai Pulmonary Hospital during January2009to December2010. The median age of the patients was47years old (ranged from12to94years old), among them,1001cases were males and431cases were females.837cases were the first-time treated patients and595cases were the repeatedly treated patients. The protocols for culture isolation for M. tuberculosis strains were as follows:1). Patient’s phlegm, other body liquids and tissue specimens were collected. After being pretreated, they were put in the isolation culture medium and cultured with BACTECTM MGITTM960Mycobacterial Detection System specific for M. tuberculosis; and2) The isolated positive cultures were smeared, stained and examined under microscopy to identify the acid-resistant M. tuberculosis, which were then inoculated into the culture tubes with medium containing PNB and TCH culture medium for strain identification; and3) The M. tuberculosis-positive bacterial inoculate were further tested for drug-sensitivity toward streptomycin, isoniazid, rifampicin, ethambutal, amikacin, capreomycin and ofloxacin.The results of drug-sensitivity test indicated:1) Out of the1432strains of M. tuberculosis,63.5%strains showed resistance to anti-tubercular drugs; and the rate of resistance to isoniazid was the highest one, accounting for46.5%; the rates of resistance to streptomycin, to rifampicin, and to ofloxacin were all over30%, and the rate of resistance to capreomycin was the lowest one, i.e. only16.1%;2) The numbers M. tuberculosis strains isolated from the repeatedly treated patients were significantly higher than those isolated from the first-time treated patients; the rate of the strains isolated from the first-time treated patients that were resistant to7drugs were all less than30%whereas rates of the strains isolated from the repeatedly treated patients that were resistant to amikacin and capreomycin were24.2%and29.2%, respectively; and their rates of resistance to other five drugs were all nearby or over50%;3) The rate of resistance to the first-line anti-tubercular drugs for all the M. tuberculosis tested was53.1%and for the first-time treated patients, it was33.8%; and for the repeatedly treated patients, it was80.2%;4) The rate of resistance to the second-line of anti-tubercular drugs for all the M. tuberculosis strains were45.8%, and for the first-time treated samples, it was33.1%and for the for the repeatedly treated samples, it was63.7%. Among the rate of resistance to the second-line anti-tubercular drugs, the rate of resistance to ofloxacin was the highest one, i.e.36.2%whereas the rate of resistance to amikacin was the lowest one, i.e.16.1%;5) Among all the M. tuberculosis strains tested, the MDR strains accounted for32.2%and XDR strains account for11.1%. Among the first-time treated patients, MDR strains accounted for10.5%and among the repeatedly treated patients, the MDR strains accounted for62,7%. The differences in the rates of MDR strains between these two groups of patients were statistically significant (p<.001). The detection rate of XDR strains among the first-time treated and the repeatedly treated patients were3.8%and21.8%, respectively. The difference in these rates between the first-treated and the repeatedly treated patients was statistically significant (p<0.001);6) For MDR strains, among all the MDR strains, XDR strains account for35.1%, and for the first-time treated patients, it accounted for36.4%whereas for the repeatedly treated patients, it accounted for34.9%. The difference in the rates of XDR strains in both the first-time treated and the repeatedly treated patients among their respective MDR stains was not statistically significant (P=0.79)(7). Among all MDR strains, pre-XDR strains accounted for42.3%, among which,93.3%were resistant to ofloxacin. Conclusion I:the rates of M. tuberculosis strains resistant to anti-tubercular drugs in Shanghai area were relatively high. Except for the lower rate of resistance to amikacin, which was below20%, the rates of resistance to a majority of anti-tubercular drugs were all higher than30%. About53.1%of the M. tuberculosis strains were resistant to the first-line anti-tubercular drugs. Among which, the rate of resistance to isoniazid was the highest one. About45.8%of M. tuberculosis strains were resistant to the second-line anti-tubercular drugs, among which, the rate of resistance to ofloxacin was the highest one.Part II:Analysis for the susceptible factors for first time-treated patients with tuberculosisThere are many factors contributing to the pathogenesis of tuberculosis, which include poverty, mental stresses, malnutrition, virus infections and the changes in host’s immunity. The interactions among these factors are very complicated. In this study, we analyzed the relationships between the parameters of the population’s nutritional status, social and environmental factors with pathogenesis of tuberculosis, aiming to provide the scientific evidence for taking the effective measures specific for the control of the epidemics of tuberculosis.We collected and performed analysis of the objectively recorded data of511cases of inpatients with confirmed tuberculosis and were first-time treated, who were admitted and treated in Shanghai Pulmonary Hospital from January2009to January2011. Among them,329cases were males and182cases were females and the median age was34years old (ranged from12to79years old). We also collected the data of the500cases of the health participants who were recruited at the same time as the control group. The information about the recruited participants included such social-environmental factors as age, sex, marital status, occupation, educational background, history of exposure to tuberculosis, the history of exposure to dust and power-like pollutants, smoking history and drinking history. The data about the parameters of such nutritional status as height, body weight, total blood proteins, albumin, triglyceride, cholesterol, high density lipoproteins, and low density lipoproteins were collected. These data were analyzed via X2test, single factor analysis, multiple factor Logistic regression analysis and investigated the susceptible factors for the patients who had confirmed tuberculosis and were first-time treated.The results of these analyses indicated that among the social-environmental factors examined, the differences in marital status (p=0.001), occupation (p<0.001), educational levels (p<0.001), and smoking history (p<0.001between the patient group and the health control groups were statistically significant. Among the nutritional parameters, the differences in body weight index (BMI)(p<0.001), total serum protein (p<0.001), albumin (p<0.001), total cholesterol (p<0.001), triglyceride (p<.001), and high density lipoproteins (p<.001) between the patient group and the health control group were statistically significant. Additionally, the multiple factor Logistic regression analysis indicated that BMI (p<.001), smoking history (p<0.001), triglyceride (p<.001), high density lipoprotein (p<.001) and low density lipoproteins (p=0.003) showed significant correlations with the pathogenesis of tuberculosis.Conclusion II:The results of this study indicated that lower educational levels, the marital status (devoiced or widowed) and smoking were the risk factors for the pathogenesis of tuberculosis. Much lower BMI and malnutrition were also the risk factors for the pathogenesis of tuberculosis. These results were similar to those reported in other countries. Moreover, this study found that high content of low density lipoproteins was a risk factor for pathogenesis of tuberculosis but the higher serum levels of triglyceride and high density lipoproteins appear to be negatively correlated with the pathogenesis of tuberculosis. This possible negative correlation needs to be verified by further study. Part Ⅲ:The relationship between the single nucleotide polymorphisms of CISH gene and susceptibility of tuberculosisIn addition to the virulent factors of M. tuberculosis, social-environmental factors and individual nutritional status, a number of studies have demonstrated that the polymorphisms of multiple genes are associated with the susceptibility of tuberculosis. For example, in2010, Khor et al. observed that the polymorphisms of CISH gene were associated with the susceptibility of a number of infectious diseases and the polymorphisms were different among different races of human populations and had different associations with the susceptibility of tuberculosis. However, there are no studies to date about the relationship between the polymorphisms of this gene and the susceptibility of tuberculosis. The aim of this study was to elucidate the relationships between the SNPs of CISH gene and the susceptibility of tuberculosis among the populations of Chinese Han nationality.We selected898cases of patients with confirmed tuberculosis who were admitted by Shanghai Pulmonary Hospital during January2009and January2011. The Multiple SNaPshot SNP typing Technology was applied to examine five locus of SNPs including rsl48685070(-639), rs414171(-292), rs6768300(-163), rs2239751(+1320) and rs622502(+3415) with DNA samples isolated from the blood samples of these patients. The blood samples were also collected from825health participants, tested and used as the control. The on-line SHEsis software in combination with the SPSS15.0statistical software were used to perform statistical analysis of the obtained results of SNPs in both patient and the control groups, including the use of Hardy-Weinberg Equilibrium Model for genetic equilibrium test. The allele frequency and genotype frequencies in a both patient group and control group were compared. The correlation between SNPs of the CISH gene and susceptibility of tuberculosis was analyzed by using single factor and multiple factors logistic regression analysis. The linkage disequilibrium analysis between different alleles was performed. The haplotype model among different SNPs was established, and its frequency was calculated. The correlation between haplotype and susceptibility of tuberculosis was analyzed.Our results indicated that:1) Among the five locus of SNPs of CISH gene examined, the locus rs148685070was only one type of allele and a genotype CC. There were no gene polymorphisms. The remaining four locus all had gene polymorphisms;2) The frequency of the locus rs414171(61.6%) within the allele A in patient group was higher than that (57.6%) of the control group, the difference was statistically significant (p<0.016). The frequency of genotype A/A (38.8%) in patient group was higher than that (31.9%) of the control group and the difference between two groups was statistically significant (p<0.011). The frequency of locus rs2239751in patient group (67.9%) was higher than that (64.5%) of the control group, the difference between two groups was statistically significant (p=0.036) whereas the difference in genotype between two groups was not statistically significant;3) The single factor analysis for the relations between SNPs and susceptibility of tuberculosis showed that age, the locus rs414171TA genotype, and locus rs2239751CC genotype were correlated with susceptibility of tuberculosis (the P values were0.017,0.003, and0.026; and the R values were1.007,0.731, and0.712, respectively);4) The multiple factor logistic repression analysis for the correlations between SNPs and susceptibility of tuberculosis indicated that age was always the independent risk factor for the pathogenesis of tuberculosis, i.e. with the increase in age, the risk of tuberculosis incidence was increased (P=0.017and R=1.008). Among the four locus of polymorphisms tested, rs2239751CC genotype and rs414171TA were the independent risk factors (p values were0.011and0.002; and the R values were0.671and0.710, respectively);5) The results of linkage disequilibrium analysis indicated that there was linkage disequilibrium between the locus of rs414171and rs2239751and between locus rs6768300and rs622502; and6) The haplotype analysis indicated that there are three haplotypes with frequency>5%, but the difference in haplotype frequency between patient group and health control group was not statistically significant.Conclusion Ⅲ:In this study, we found that SNPs of CISH gene were correlated with susceptibility of tuberculosis. Among the4locus of polymorphisms examined, the locus rs2239751CC genotype and rs414171TA genotype were the independent risk factors. There are race differences in the relations between polymorphisms of CISH gene and susceptibility of tuberculosis. This is the first study on the analysis of relationships between the single nucleotide polymorphisms of CISH genes and susceptibility of tuberculosis among population of Chinese Han nationality. |
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