Table of Content

10 June 2014, Volume 36 Issue 6

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Strengthen basic research capacity on tuberculosis and promote the clinical diagnosis and treatment

PANG Yu，ZHAO Yan-lin

Chinese Journal of Antituberculosis. 2014, 36(6):  409-410.  doi:10.3969/j.issn.1000-6621.2014.06.001

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Analysis of the relativity between the drug resistance and genotype of clinical strains isolated from tuberculous meningitis

WANG Ting, ZHAO Yan-lin, PANG Yu, ZHOU Yang, FENG Guo-dong, PENG Jin-xiang, ZHAO Gang

Chinese Journal of Antituberculosis. 2014, 36(6):  411-417.  doi:10.3969/j.issn.1000-6621.2014.06.002

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Objective To study the relativity between the drug resistance and genotype of clinical strains isolated from tuberculous meningitis(TBM).  Methods M. tuberculosis was cultured and isolated in cerebrospinal fluids (CSF) from 25 cases diagnosed with TBM in Xijing Hospital. These isolates were performed the susceptibility testing of 14 antituberculosis drugs, the sequencing of 10 drug-resistant genes and strain typing by Spoligotyping.  Results Of 25 M. tuberculosis isolates, 20 (80.0%) clinical isolates were identified as Beijing genotype by Spoligotyping; 3 (3/25) strains were multidrug-resistant (MDR) and belonged to Beijing genotype, in which katG, rpoB, gyrA, rrs-KAN, rpsL, gidB genes were found the mutations, but inhA, embB, gyrB, eis genes had not mutation by the DNA sequencing.  Conclusion Beijing genotype strains may act as the main epidemic strain of TBM，especially MDR-TBM.  

Study on the relationship between single nucleotide polymorphisms of IFNA8 gene and susceptibility to Mycobacterium tuberculosis in Han Chinese

WEN Yu-xin, ZHANG Guo-liang, LIN Qiao, ZHONG Hong-jian, ZHANG Ming-xia, YANG Lin, CHEN Xin-chun, WANG Zheng

Chinese Journal of Antituberculosis. 2014, 36(6):  418-423.  doi:10.3969/j.issn.1000-6621.2014.06.003

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Objective To study the association between single nucleotide polymorphisms (SNP) of IFNA8 gene and the susceptibility to Mycobacterium tuberculosis in Han Chinese. Methods From January 2010 to December 2011, clinical data of pulmonary tuberculosis patients was collected from Shenzhen Third People’s Hospital. There were 1533 patients with tuberculosis as case group and 1445 volunteers as healthy controls. A case-control study was used in the research. SNPs of IFNA8 (rs1330322，rs10964982，rs4978116) were typed by a high-throughput sequenom genotyping platform. The allele frequency (AF), odds ratio (OR) and 95% confidence interval (95%CI) were calculated between case group and control group by using Chi-square test, P<0.05 was consi-dered to be significant. Results (1) The frequencies of allele A in the genetic locus rs1330322 were 30.5%(936/3066) in tuberculosis patients (TB) group and 27.8%(804/2890) in healthy control (HC) group. There were statistical differences between two groups(χ²=5.277, OR=1.140, 95%CI=1.019－1.275, P=0.022). The minor allele frequencies of rs10964982 and rs4978116 of TB group were 17.0% （520/3066） and 28.8%（882/3066）, respectively. And the minor allele frequencies of rs10964982 and rs4978116 of HC group were 17.0%(491/2890) and 28.6%(826/2890), respectively. The minor allele frequencies of rs10964982 and rs4978116 were no significant differences between two groups (χ²=0.001 and 0.025，P=0.976 and 0.874). （2） In sexual stratified analysis of rs1330322, the frequencies of allele A in female population of case group were 30.8%（330/1072）, and 26.6%（300/1126） in female of HC group. There were significant differences between two groups of female population (χ²=4.605，OR=1.225，95%CI=1.018－1.474，P=0.032). However, among male population, the frequencies of allele A were 30.4%（606/1994） and 28.6%(504/1764) in TB group and HC group without significant differences statistically (χ²=1.489, OR=1.091, 95%CI=0.948－1.256，P=0.222). （3） The frequencies of allele A in female less than 25 years old were 33.8%（111/328）and 25.2% （112/444）, respectively. There were significant differences between TB group and HC group (χ²=6.818，OR=1.516，95%CI=1.108－2.074，P=0.009). But the frequencies of allele A in female more than 25 years old were not significantly different between two groups (χ²=0.610，OR=1.096，95%CI=0.871－1.380，P=0.435).  Conclusion The genetic polymorphism of IFNA8 (rs1330322) is associated with TB, especially female population less than 25 years old. Allele A may be a risk factor for prediction of TB in female less than 25 years old.

Immunogenicity of different dosage DNA vaccine from Mycobacterium tuberculosis medicated by electroporation

LIANG Yan, XIAO Li, BAI Xue-juan, GAO Yu, YANG You-rong, ZHANG Xiao-yan, CHEN Dan, WANG Lan, SHI Ying-chang, ZHANG Jun-xian, LI Zhong-ming, WU Xue-qiong

Chinese Journal of Antituberculosis. 2014, 36(6):  424-428.  doi:10.3969/j.issn.1000-6621.2014.06.004

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Objective To study the immunogenicity of different dosages DNA vaccines from Mycobacterium tuberculosis medicated by electroporation.  Methods Twenty female BALB/c mice were immunized intramuscularly with saline, 10 μg Ag85A DNA, 50 μg Ag85A DNA and 100 μg Ag85A DNA for three times at two-week intervals, respectively. Twenty female BALB/c mice were medicated intramuscularly by electroporation with saline, 10 μg Ag85A DNA, 50 μg Ag85A DNA and 100 μg Ag85A DNA for 3 times at two-week intervals, respectively. There were 5 mice each group. Mice were sacrificed at 2 weeks after the final immunization respectively. The levels of IFN-γ and IL-4 in the culture supernatants of splenic lymphocytes were measured with enzyme-linked immunosorbent assay (ELISA). The ratio of CD4⁺ T cells expressing IFN-γ (Th1) and IL-4 (Th2) in whole blood was detected by flow cytometry. Data were expressed as means and standard deviations. Statistical significance between each group was calculated using two factors factorial design ANOVA followed by compared with t test using SAS 9.2 software and a P-value of <0.05 was considered to be statistically significant.  Results At 2 weeks after the final immunization, IFN-γ levels in splenocyte culture supernatant in 50 μg DNA (646.05±342.53) pg/ml and 100 μg DNA intramuscular injection group (738.61±372.68) pg/ml was significantly higher than that in saline (1.73±3.88) pg/ml (t=4.065 and 4.647, all P<0.05) and 10 μg DNA intramuscular injection group (87.83±120.82) pg/ml (t=3.513 and 4.094, all P<0.05); IFN-γ level in splenocyte culture supernatant in 10 μg DNA electroporation group (357.06±105.18) pg/ml was significantly higher than that in saline (t=2.247, P<0.05)，and higher than that in 100 μg DNA electroporation group (86.08±135.73) pg/ml，but the difference was not statistically significant(t=1.706, P>0.05). 50 μg DNA electroporation group (648.60±439.41) pg/ml was significantly higher than that in saline(t=4.081, P<0.05) and 100 μg DNA electroporation group(t=3.539, P<0.05); compared with intramuscular injection group, IFN-γ levels of 10 μg DNA electroporation group increased 3 times (357.06 pg/ml)/(87.83 pg/ml); that of 50 μg DNA electroporation group had no significant change (t=－0.016, P＞0.05); that in 100 μg DNA electroporation group declined 88.35% (t=－4.105, P<0.05). Percentage of Th1 cells secreted IFN-γ in different dosages of intramuscular DNA (10 μg Ag85A DNA：(1.39±0.84)%, 50 μg Ag85A DNA：(1.55±0.33)%, 100 μg Ag85A DNA：(2.13±0.47)%) and DNA electroporation groups (10 μg Ag85A DNA：(1.42±0.47)%, 50 μg Ag85A DNA：(1.88±0.51)%, 100 μg Ag85A DNA：(1.43±0.68)%) were higher than that in saline group (0.65±0.31)% (t=2.002, 2.431, 4.015, 2.084, 3.332 and 2.105, all P<0.05), there has no statistical difference between doages DNA electroporation groups and intramuscular injection groups (t=0.081, 0.901 and －1.91, all P>0.05). Percentage of Th2 cells secreted IL-4 in different dosages of intramuscular DNA (10 μg Ag85A DNA：(1.42±1.18)%, 50 μg Ag85A DNA：(1.14±0.78)%, 100 μg Ag85A DNA：(1.24±0.76)%) and DNA electroporation groups (10 μg Ag85A DNA：(1.19±1.09)%, 50 μg Ag85A DNA：(2.06±0.96)%, 100 μg Ag85A DNA：(1.47±0.65)%) were higher than that in saline electroporation group (4.14±2.55)% (t=－3.392, －3.738, －3.616, －3.676, －2.599 and －3.325, all P<0.05), there was no statistically significant difference between doages DNA electroporation groups and intramuscular injection groups(t=0.284, －1.139 and －0.292, all P>0.05).  Conclusion The results suggest that lower doses of DNA immunization by electroporation could improve immune response, using a small amount of DNA vaccine can produce good immune effect.

Correlation between the mutations of gyrase gene and fluoroquinolone resistance in Mycobacterium tuberculosis

LU Feng-yue, YU Ri-xia, HU Zu-qiong, CAI Xing-shan, TAN Yao-ju

Chinese Journal of Antituberculosis. 2014, 36(6):  429-433.  doi:10.3969/j.issn.1000-6621.2014.06.005

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Objective To analyze the mutations of gyrase (gyr) gene in multidrug-resistant (MDR) M.tuberculosis clinical isolates, and to investigate the correlation between the gyr mutations and fluoroquinolone (FQ) resistance.  Methods Seventeen MDR M.tuberculosis isolates were analyzed the FQ resistance using minimum inhi-bitory concentration (MIC) method, and detected gyr mutations by DNA sequencing.  Results No mutation was observed in gyrA and gyrB genes of M.tuberculosis H37Rv, while 17 clinical MDR isolates all carried the substitution AGC→ACC at codon 95. Among 12 FQ-resistant isolates, 10 (83.3%) harbored missense mutation in gyrA gene, including codon 89, 90, 91 and 94. In addition, 1 isolate had mutation at codon 500 of gyrB gene. Among 9 moxifloxacin-resistant isolates, 8 (88.9%) showed the mutations in gyrA gene.  Conclusion The mutation in gyrA is one of the most important mechanism for FQ resistance in M. tuberculosis. The most frequent mutations located at codon 89, 90, 91 and 94.  

Methodological comparison of MALDI-TOF and TaqMan probe technology in SNP genotyping and their application in screening SNP with susceptibility to tuberculosis

WANG Wen-fei, ZHANG Guo-liang, YANG Fan, YANG Hui, ZHAO Li-fang, XU Fa-sheng, ZHANG Ming-xia, CHEN Xin-chun

Chinese Journal of Antituberculosis. 2014, 36(6):  434-439.  doi:10.3969/j.issn.1000-6621.2014.06.006

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Objective To develop a new method based on MALDI-TOF and TaqMan probe for screening the single nucleotide polymorphisms (SNP) with susceptibility to tuberculosis and evaluate its clinical application value. Methods At first, total 7 sites we targeted were detected by MALDI-TOF simultaneously. The single SNP site was detected by using TaqMan probe technology. The two methods were compared by accuracy and sensitivity. We determined a susceptibility gene site by comparing genotype and gene frequency between 400 TB patients and 300 healthy controls by using this method.  Results The success rate of genotyping by MALDI-TOF was 99.7% （698/700）, that of TaqMan probe technology was 98.4% （689/700）． The frequency of G allele at rs2227473 in patient groups (90.3%, 722/800) was significantly higher than that in control groups (82.5%, 495/600) (χ²=6.911, P=0.009)．The other 6 SNP sites had no significant difference between two groups.  Conclusion We successfully established a new method for screening the SNPs with susceptibility to tuberculosis. The polymorphism of IL-22 rs2227473 allele G may be associated with the susceptibility to pulmonary tuberculosis, allele A may be the protective gene.

MF59 adjuvant enhances immunogenicity of heat-killed BCG

HUANG Xiang-yu, ZHANG Chun-qing, LI Jun-li, SHAO Jin-shi, HE Xiu-yun

Chinese Journal of Antituberculosis. 2014, 36(6):  440-446.  doi:10.3969/j.issn.1000-6621.2014.06.007

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Objective To study the effects of MF59 (an oil-in-water emulsion composed of small droplets of squalene surrounded by a monolayer of nonionic detergents (Span85 and Tween 80)) as adjuvant on immune responses to the heat-killed Bacillus Calmette-Guérin (hBCG) to prove MF59 enhancing the cellular immune responses to hBCG. Methods BALB/c mice were divided into seven groups, which were immunized with MF59 (group A), low doses of hBCG (0.025 mg/ml） (group B), middle doses of hBCG (0.25 mg/ml) (group C), high doses of hBCG (2.5 mg/ml) (group D), MF59+ low doses of hBCG (group E), MF59+ middle doses of hBCG (group F) and MF59+ high doses of hBCG (group G) for three times at intervals of two weeks, respectively. The mice were sacrificed two weeks after the last immunization. The splenic lymphocytes and peritoneal macrophages were isolated and cultured with BCG-PPD. Sandwich ELISA and ELISPOT assay were used to detect BCG-PPD-specific cytokines in culture supernatants and the number of IFN-γ, IL-2, IL-4 producing spot forming cells (SFCs), respectively. One-way analysis of variance with least-significant-difference (LSD) comparisons was used to test differences among the levels of cytokines and the number of SFCs. P values of <0.05 were considered statistically significant. Results The number of IFN-γ, IL-2, IL-4 producing SFCs in group G (151.3±66.6, 247.8±58.0, and 65.8±24.6) was higher than that in other groups (the biggest means of IFN-γ, IL-2, IL-4 producing SFCs were (30.4±13.0), (37.1±10.8), and (16.4±9.7), respectively) (t=3.2, P=0.007 for IFN-γ producing SFCs；t=3.6, P=0.003 for IL-2 producing SFCs；t=3.0, P=0.01 for IL-4 producing SFCs). The levels of IL-1β in the supernatants from BCG-PPD-stimulated peritoneal macrophages were higher in groups E (663.3±177.5 pg/ml), F (813.3±193.4 pg/ml), and G (742.3±316.1 pg/ml) than that in groups A (104.7±65.8 pg/ml), B (82.9±54.8 pg/ml) and C (66.5±53.5 pg/ml) (group E vs A, t=2.9, P=0.01; group F vs A, t=3.5, P=0.004; group G vs A, t=2.5, P=0.031; group E vs B, t=3.1, P=0.007; group F vs B, t=3.6, P=0.003; group G vs B, t=2.6, P=0.024; group E vs C, t=3.0, P=0.01；group F vs C, t=3.5, P=0.004；group G vs C, t=2.5, P=0.031). The levels of IL-12 were lower in groups A (36.3±20.5 pg/ml), C (94.5±20.6 pg/ml) and G (128.2±54.6 pg/ml) than that in groups E (545.7±97.2 pg/ml) and F (665.5±295.4 pg/ml) (group A vs E, t=－5.1, P=0.000; group C vs E, t=－4.5, P=0.000; group G vs E, t=－3.7, P=0.002; group A vs F, t=－4.4, P=0.001; group C vs F, t=－3.7, P=0.002; group G vs F, t=－2.9, P=0.012). Conclusion MF59 combined with high dose of hBCG adjuvant can induce BCG-PPD specific IFN-γ, IL-2, and IL-4 secretion from splenic lymphocytes, and MF59 combined with low or middle doses of hBCG adjuvant can increase the levels of IL-1β and IL-12 secreted from the macrophage.

Rapid detection of Mycobacterium bovis for the diagnosis of tuberculosis in dairy cattle

JIANG Yuan, WANG Qu-zhi, ZHANG Yang-yi, SHEN Su-fang, LI Jing, TANG Wen-hong, GUI Xiao-hong, MEI Jian, PAN Qi-chao, SHEN Xin

Chinese Journal of Antituberculosis. 2014, 36(6):  447-452.  doi:10.3969/j.issn.1000-6621.2014.06.008

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Objective To evaluate a rapid diagnosis of tuberculosis among dairy cattles using a MGIT 960 system, and to provide a laboratory data for improving dairy cattle tuberculosis quarantine. Methods  Nine thousand three hundred and fifty-five dairy cattles were detected by tuberculin-test (PPD) from 5 different dairy farms in Shanghai during 2010 to 2012, and 223 dairy cattles were detected as PPD (+). Among those 223 PPD (+) cattles, 49 cattles were selected randomly using the random number table. A total of 98 lung lymph node tissue specimens were collected from these 49 PPD (+) cattles. Culture and identification of Mycobacterium for the 98 lymph node tissue specimens were performed by MGIT 960 methods, chromatographic immunoassay, PCR-based genomic deletion analysis and DNA sequencing.  Results A total of 8 dairy cattles were diagnosed as Mycobacterium among which 1 cattle was identified as M.bovis, 6 cattles were identified as M.avium and 1 cattle was identified as M.nonchromogenicum.  Conclusion In order to improve the specificity of detection for bovine tuberculosis, we recommend that culture and species identification by using rapid detection techniques should be introduced into the laboratory diagnosis as a supplement for tuberculin-test.

Study on the cross-resistance among 5 different fluroquinolones in ofloxacin-resistant Mycobacterium tuberculosis isolates

WANG Qian, SONG Yuan-yuan, PANG Yu, WANG Yu-feng, OUYANG Qin, ZHAO Yan-lin

Chinese Journal of Antituberculosis. 2014, 36(6):  453-457.  doi:10.3969/j.issn.1000-6621.2014.06.009

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Objective To investigate the cross-resistance among the different fluroquinolones (FQs) in oflo-xacin-resistant Mycobacterium tuberculosis(M. tuberculosis) isolates.  Methods One hundred and thirty ofloxacin-resistant M. tuberculosis isolates were selected from national tuberculosis drug-resistant survey conducted in 2007. Broth dilution method was used to detect the minimal inhibitory concentration (MIC) of M. tuberculosis against five FQs, including ofloxacin (Ofx), levofloxacin (Lfx), moxifloxacin (Mfx), gatifloxacin (Gfx) and sparfloxacin (Sfx). Chi square test was performed to compared the drug resistant rates of different FQs. The difference was considered as significant when P<0.05.  Results Of 130 Ofx-resistant M. tuberculosis isolates, there were 110 (84.6%，110/130), 108 (83.1%，108/130), 46 (35.4%，46/130) and 66 (50.8%，66/130) isolates resistant to Lfx, Mfx, Gfx and Sfx, respectively. Statistical analysis revealed that the Gfx-resistant rate of M. tuberculosis isolates was significantly lower than those of Lfx（χ²=65.641, P<0.01）, Mfx（χ²=61.225, P<0.01） and Sfx（χ²=6.274, P=0.02）. Among the Lfx-resistant isolates, 90.9%（100/110）, 41.8%（46/110） and 59.1%（65/110） were resistant to Mfx, Gfx and Sfx, respectively. In addition, there were 97.8% (45/46) and 97.0% (64/66) of Gfx- and Sfx-resistant isolates resistant to Mfx, respectively. For Gfx- and Sfx-resistant isolates, only 41.7%（45/108） and 59.3%（64/108） showed resistance to Mfx, respectively.  Conclusion  Our findings demonstrate that the Gfx- and Sfx-resistant rates were significantly lower than those of the others. In addition, the cross-resistance is observed between Lfx and Mfx, while there is partially unidirectional cross-resistance between Mfx and Gfx/Sfx.

Evaluation of the simultaneous amplification and testing for diagnosis of Mycobacterium tuberculosis

CAI Xing-shan, MA Pin-yun, ZHANG Yuan-liang, TAN Yao-ju

Chinese Journal of Antituberculosis. 2014, 36(6):  458-461.  doi:10.3969/j.issn.1000-6621.2014.06.010

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Objective To assess the clinical value of the isothermal RNA amplification assay (SAT) for detection of Mycobacterium tuberculosis in sputum, body fluid and nidus samples． Methods Seven hundred and thirty-four sputum samples, 94 samples of body fluid (including 68 thoracic effusion, 21 cerebrospinal fluid and 5 joint effusion) and 76 nidus samples collected from suspected tuberculosis patients were tested by both SAT and L-J culture. The positive isolates were identified by Gene-chip method. The samples with different results between SAT and L-J culture were tested by Mycobacterium tuberculosis PCR (PCR-TB) fluorescence diagnosis kits．The detection rates of SAT and L-J culture were analyzed by Chi-square test.  Results With the result of L-J culture as the re-ference, the sensitivity, the specificity and the coincidence rate of the sputum samples were 90.20% (30/255), 92.48% (443/479) and 91.69% (673/734), those of the fluid samples were 94.74% (18/19), 81.33% (61/75) and 84.04% (79/94), and those of the nidus samples were 100.00% (14/14), 77.42% (48/62) and 81.58% (62/76). With the result of PCR-TB as the reference, the sensitivity, the specificity and the accordance rate of the sputum samples were 96.30% (260/270), 99.35% (461/464) and 98.23% (721/734), those of the fluid samples were 96.97% (32/33), 100.00% (61/61) and 98.94% (93/94), and those of the nidus samples were 100.00% (27/27), 97.96% (48/49) and 98.68% (75/76).The detection rates the three different kinds of specimens by L-J culture and SAT were 34.74% (255/734) vs 36.24% (266/734), 20.21% (19/94) vs 34.04% (32/94), 18.42% (14/76) vs 36.84%(28/76). There was no difference in sputum samples between SAT and L-J culture (χ²=0.36, P＞0.05). However, there were significant differences in body fluids and nidus samples between SAT and L-J culture (χ²=4.55 and 6.44, all P<0.05).  Conclusion  SAT is a rapid, sensitive and specific method for detection of Mycobacterium tuberculosis in kinds of clinical samples.

Application and evaluation of Xpert Mtb/RIF detection assay in the diagnosis of tuberculosis

ZHAO Bing, OU Xi-chao, XIA Hui, LI Qiang, PANG Yu, ZHANG Zhi-ying, DONG Hai-yan, LI Jun-chen, ZHANG Jian-kang, CHI Jun-ying, ZHAO Yan-lin

Chinese Journal of Antituberculosis. 2014, 36(6):  462-466.  doi:10.3969/j.issn.1000-6621.2014.06.011

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Objective To evaluate the application value of Xpert Mtb/RIF detection assay in the diagnoses of tuberculosis (TB) and Rifampin resistance. Methods Clinicians continuously recruited 2142 TB suspects in four counties (districts) level TB dispensary (Xiangtan, Yueyang, Beilin, Lanxi). Three sputum specimens each patient provided were performed smear, solid culture and Xpert Mtb/RIF test. The TB patients with definite diagnoses were followed up for two months. The staff in provincial laboratory completed rifampin susceptibility test in 555 culture-positive strains using proportional method. The staff in national reference laboratory sequenced the rifampin-resistant determinant region of rpoB genes in the strains with the inconsistent results between Xpert Mtb/RIF and traditional drug susceptibility test. Using clinical diagnosis as gold standard, the performance of smear, solid culture and Mtb/RIF test were evaluated. The performance of Xpert Mtb/RIF for rifampin resistance detection was compared with traditional drug susceptibility test.   Results Using clinical diagnosis result as the gold standard, the sensitivities of smear, solid culture and Xpert Mtb/RIF were 25.68%（284/1106）, 51.44%（555/1079） and 58.82%（650/1105）, respectively. The sensitivity of Xpert Mtb/RIF was higher than smear (χ²=360.10, P<0.05) and the solid culture(χ²=50.13, P<0.05). Compared with traditional drug susceptibility test, the sensitivity and the specificity of Xpert Mtb/RIF for rifampin resistance detection were 87.10% (27/31) and 97.95% (477/487), respectively. Conclusion Xpert Mtb/RIF test is simple, the sensitivity of Xpert Mtb/RIF is higher than smear microscopy and solid culture，it can also diagnose patients of rifampin resistance, so it has very good application prospects in our county (district) level laboratories.

Adjunctive diagnostic value of interferon-gamma release assay in tuberculosis lymphadenitis

JIA Hong-yan, PAN Li-ping, LIU Fei, DU Bo-ping, SUN Qi, XING Ai-ying, DU Feng-jiao, MA Yu, ZHANG Zong-de

Chinese Journal of Antituberculosis. 2014, 36(6):  467-471.  doi:10.3969/j.issn.1000-6621.2014.06.012

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Objective To evaluate the value of interferon-gamma release assay (T-SPOT.TB) in the diagnosis of tuberculosis lymphadenitis. Methods From July 2011 to Dec 2013, 185 patients with enlarged lymph node were enrolled and categorized as histopathology-confirmed tuberculosis lymphadenitis group (n=51) and no-tuberculosis lymphadenitis group (n=105). The remaining inconclusive patients were excluded from final analysis. T-SPOT.TB with peripheral blood and tuberculosis antibody with serum by using western blot method were detected in all patients. Statistical analysis was performed using SPSS 17.0. The measurement data of non normal distribution were expressed with the median. Spot forming cells(SFCs) were compared in different groups using nonparametric Mann Whitney U test. Comparisons between proportions were performed using Chi-squared test. Results The sensitivity and specificity of T-SPOT.TB assay were 92.2％(47/51) and 79.0％(83/105) respectively, however, they were 60.8％(31/51) and 77.1％(81/105) in tuberculosis antibody, respectively. The sensitivity of T-SPOT.TB assay was significantly higher than that of tuberculosis antibody with statistically significant (χ² =13.95，P<0.01). Also, SFCs median of tuberculosis lymphadenitis group and non-tuberculosis lymphadenitis group were 242(57, 621)/ 10⁶ PBMCs and 0(0, 20) /10⁶ PBMCs, respectively. SFCs was significantly higher in the tuberculosis lymphadenitis group than that in non-tuberculosis lymphadenitis group (U=472.0，P<0.01). In the 69 patients with positive T-SPOT.TB results, SFCs median with 280（98，684）/10⁶ PBMCs in the 47 patients with tuberculosis lymphadenitis was significantly higher than 52(35，93）/10⁶ PBMCs in the 22 patients with non-tuberculosis lymphadenitis (U=146.5，P<0.01). Conclusion The T-SPOT.TB assay is high sensitivity in the diagnosis of tuberculosis lymphadenitis. It could be a useful adjunctive tool for diagnosis of tuberculosis lymphadenitis.

Evaluation of GeneXpert Mtb/RIF assay for diagnosis of tuberculosis and rifampin-resistant tuberculosis

LI Hui, TAN Yao-ju, LI Hong-min, MA Xiao-guang, XING Jin, HAO Bao-lin, ZHAO Yan-lin

Chinese Journal of Antituberculosis. 2014, 36(6):  472-476.  doi:10.3969/j.issn.1000-6621.2014.06.013

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Objective To evaluate the performance of GeneXpert Mtb/RIF assay for the diagnosis of Mycobacterium tuberculosis (Mtb) and rifampin-resistant tuberculosis (TB) in clinical samples. Methods The sputum samples were collected from all TB suspects who visited the outpatient departments of the 309th Hospital of Chinese People’s Liberation Army and Guangzhou Chest Hospital, as well as the TB clinic of He’nan Provincial Center for Disease Control and Prevention (He’nan Provincial CDC) from January to May 2011. Smear microscopy, culture (golden-standard method), drug susceptibility testing (DST, conventional proportion method) and GeneXpert Mtb/RIF assay were respectively performed on each clinical sample. SPSS software version 11.5 was used for statistical analysis to compare the diagnostic sensitivity and specificity of GeneXpert Mtb/RIF assay and other examination methods, P<0.05 was regarded as a significant difference. Results Sputum samples were collected from 1971 TB suspects. The samples from 3 suspected cases were contaminated and were excluded from the analysis. Thus 1968 suspected cases were involved in the analysis. Compared with the results of smear microscopy and culture, the sensitivity and specificity of GeneXpert Mtb/RIF assay for diagnosis of TB was 93.27%(748/802), 91.93%(797/867) and 91.60%(1068/1166), 95.55%(1052/1101) respectively; compared with the results of clinical diagnosis, the sensitivity and specificity of GeneXpert Mtb/RIF assay for diagnosis of TB was 78.66%(822/1045) and 98.67%(888/900) respectively; compared with the results of conventional DST, the sensitivity and specificity of GeneXpert Mtb/RIF assay for diagnosis of rifampin-resistant TB was 97.92%(188/192) and 100.00%(595/595) respectively. Conclusion GeneXpert Mtb/RIF assay is a rapid, simple and hand-free molecular diagnostic tool with high sensitivity and specificity for the diagnosis of TB and rifampin-resistant TB. It can be used for clinical diagnosis of TB and rifampin-resistant TB.

The value of interferon-gamma release assays for the differential diagnosis between pulmonary tuberculosis and lung disease of non-tuberculosis mycobacteria (NTM)

LIN Ling，LIU Hou-ming，DENG Qun-yi，ZHANG Jie-yun，ZHANG Ming-xia，ZHU Xiu-yun，ZHANG Guo-liang，DENG Guo-fang，YUE Jian-rong，CHEN Xin-chun，XIE Hong

Chinese Journal of Antituberculosis. 2014, 36(6):  477-481.  doi:10.3969/j.issn.1000-6621.2014.06.014

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Objective To evaluate the value of interferon gamma release assays for the differential diagnosis between pulmonary tuberculosis and lung disease of non-tuberculous mycobacteria (NTM).  Methods We chose 334 cases of pulmonary disease admitted in the Third People’s Hospital of Shenzhen during the period from Februa-ry 2011 to May 2013. Three hundred and thirty-four sputum specimens from these patients were identified by PCR-reverse dot blot hybridization assay. An in-house ELISPOT assay was used to determine Mycobacterium tuberculosis antigen-specific IFN-γ production in patients; PCR-fluorescent probe was used to detect Mycobacterium tuberculosis DNA in sputum specimens (fluorescent quantitative PCR).   Results Of 334 cases, 240 cases were identified as Mycobacterium tuberculosis infection and 94 were identified as non-tuberculosis mycobacteria infection. The positive rate of fluorescent quantitative PCR and IGRAs were 74.58%（179/240）and 77.08%（185/240）, respectively in patients with PTB,while 10.64%（10/94） and 20.21%（19/94）,respectively in patients with lung disease of non-tuberculosis mycobacteria. The sensitivity of the fluorescent quantitative PCR and IGRAs were 74.58%（179/240）and 77.08%（185/240）, respectively; while the specificity of the two methods were 89.36%（84/94）and 79.79%（75/94）, respectively. The sensitivity and specificity of two methods combined were 93.75%（225/240）and 77.66%（73/94）, respectively.  Conclusion IGRAs can detect rapidly and differentiate pulmonary tuberculosis from lung disease of non-tuberculosis mycobacteria, and the accuracy of differential diagnosis can be improved significantly when IGRAs combined with PCR, which is important to select antituberculosis drugs for patients.

Down-regulation of IFNAR gene in macrophage induced by Mycobacterium tuberculosis and its role in peripheral bloods of patients with tuberculosis

ZHANG Guo-liang，ZHAN Sen-lin，ZHONG Hong-jian，LIN Qiao，WANG Wen-fei，JIN Xiao-fei，ZHANG Ming-xia，CHEN Xin-chun

Chinese Journal of Antituberculosis. 2014, 36(6):  482-486.  doi:10.3969/j.issn.1000-6621.2014.06.015

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Objective To explore whether Mycobacterium tuberculosis (Mtb) regulates the expression of IFNAR gene and its role in the patients with tuberculosis (TB). Methods The cases were selected from Shenzhen Third People’s Hospital during January to May 2013，involving sputum smear-positive TB patients（n=15），the persons with latent tuberculosis infection (LTBI) (n=15) and healthy controls (HC) (n=15). CD14⁺ monocytes were isolated from peripheral blood mononuclear cells (PBMCs) and differentiated into human macrophages under macrophage colony stimulatory factor (M-CSF) stimulation，then expression of IFNAR1 and IFNAR2 genes was determined with qPCR assay after infection with Mtb H37Ra or H37Rv strains. All data was dealt with GraphPad 4.0 software. The homogeneity test of variances was used to judge whether multiple measurement data were in Gaussian distribution，if so，the data was shown as x±s and analysis of variance（ANOVA）was used to compare multiple comparison to calculate q value. Enumeration data was compared with R×C Chi-square test. It was considered as signifi-cant difference with P<0.05. Results IFNAR1 gene expression in the macrophages infected by Mtb H37Rv decreased significantly compared to the blank control (5.95±0.41 vs 7.53±0.41，q=4.15，P<0.05). IFNAR1 gene expression in the macrophages infected by Mtb H37Ra（6.54±0.33） had not significant difference with the blank control （7.53±0.41）and H37Rv infection（5.95±0.41）（q=2.56 and 1.57，P>0.05）. IFNAR2 gene expression had not significant difference in the macrophages infected by Mtb H37Rv （8.81±0.85）or H37Ra （8.84±0.80）（q=0.02，P>0.05）, but they were significantly lower compared to the blank control（12.67±1.15）（q=4.10 and 4.13，P<0.05）. Furthermore， IFNAR1 gene expression had no significant difference among different groups（TB 5.39±0.64；LTBI 6.59±0.86；HC 7.08±1.10，P>0.05），and IFNAR2 gene expression was lower in TB than that in HC（5.96±0.58 vs 10.01±1.58，q=3.67，P<0.05）. Conclusion Mycobacterium tuberculosis could induce down-regulation of IFNAR gene expression in the macrophages, which was not associated with virulence of Mtb. IFNAR2 gene expression was lower in TB and could be considered as a new diagnosis marker.

Analysis of the results of mycobacteria susceptibility test in cerebrospinal fluid and CD4⁺T lymphocyte counts in peripheral blood from 95 cases with tuberculous meningitis

LI Tong-xin, ZHONG Min, FU Xiao, DING Xian-ping

Chinese Journal of Antituberculosis. 2014, 36(6):  487-493.  doi:10.3969/j.issn.1000-6621.2014.06.016

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Objective To retrospectively analyze drug susceptibility test and strain identification of Mycobacterium tuberculosis in cerebrospinal fluid, as well as investigate the results of CD4⁺ T lymphocyte counts in peri-pheral blood from the patients with tuberculous meningitis (TBM), to provide a basis for early intervention of tuberculous meningitis. Methods Clinical data of 95 cases with the recorded drug susceptibility test of Mycobacterium tuberculosis were collected from tuberculous meningitis department of Public Health Medical Center on Chongqing during Jan. 3, 2011 to Nov. 27, 2013, in which there were 52 male and 43 female; 73 cases of initial treatment, 21 cases of retreatment (excluding 1 case infected with non-tuberculosis mycobacterium); 16 cases with positive HIV antibody by clinical blood detection. The Chi-square test was used to analyze the results of drug susceptibility test (including total 11 kinds of drugs: streptomycin (Sm), isoniazid (INH), rifampicin (RFP), ethambutol (EMB), amikacin, ofloxacin, capreomycin, ethionamide, pasiniazide (PAIN), rifapentine (Rft), sodium aminosalicylate), strain identification, and the counts of CD4⁺ T lymphocyte in peripheral blood in the early stage of treatment. Resistance characteristics and the influencing factors of either resistance or count were analyzed, and when P-values <0.05, the difference between two means was considered statistically significant.  Results (1)The drug susceptibility test and species identification: 1 case (1/95, 1.1％) of non-tuberculosis mycobacterium was resistant to 11 kinds of anti-tuberculosis drugs; Of 94 cases (94/95, 98.9％) of Mycobacterium tuberculosis complex, 40 cases (42.6%) had varying degrees of resistance to 11 kinds of anti-tuberculosis drugs, MDR-TB was 12.8% (12/94), and XDR-TB was 1.1% (1/94). (2)Drug-resistant types: 9.6％(9/94) cases were resistant to single drug; 16.0％(15/94) cases were resistant to two kinds of drugs; 2.1% (2/94) cases were resistant to three kinds of drugs, and 14.9% (14/94) were resistant to more than three kinds of drugs. (3)The top six rank of drug resistances: Sm 26.6% (25/94)＞INH 23.4% (22/94)＞RFP 18.1% (17/94)＞PAIN 16.0% (15/94)＞EMB and Rft 14.9%(14/94). (4) The counts of CD4⁺ T lymphocytes in peripheral bloods from 11 cases (11/94, 11.7％) with TBM were within the normal range. The gender (male:90.2%(46/51), female:86.0%（37/43）), initial treatment 90.4% (66/73) or retreatment 81.0% (17/21) did not effect on the count (χ²-values were 7.76, 7.44 respectively, P＞0.05). The counts of CD4⁺ T lymphocytes in the cases with HIV antibody-positive TBM 100.0%(16/16) were significantly lower than those with HIV antibody-negative TBM 85.9%(67/78) (χ²-value was 34.21, P<0.01). Conclusion Ninety-four TBM cases with either initial treatment or retreatment showed high drug-resistant trend, and their counts of CD4⁺ T lymphocyte in peripheral blood were generally low, especially the TBM cases complicated with AIDS.

Clinical study on local drug perfusion via bronchoscope in the treatment of bronchial tuberculosis

TIAN Jiang-hua, DAI Yuan-rong, YAN Sun-shun, LIN Jie, WENG Hai-xia, WU Li-qin, XIA Xiao-dong, WU Cheng-yun, CHEN Ye

Chinese Journal of Antituberculosis. 2014, 36(6):  494-497.  doi:10.3969/j.issn.1000-6621.2014.06.017

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Objective To explore the clinical efficacy and value of the local infusion of anti-tuberculosis (TB) drugs by bronchoscopy combined with anti-TB chemotherapy in the treatment of tracheobronchial TB.  Methods Sixty-two patients with tracheobronchial TB were enrolled in this study and randomly divided into two groups by using the method of random number table: the “combined treatment group”, in which the patients received both local infusion and chemotherapy of anti-TB drugs; and the “simple chemotherapy group” or “control group”, in which patients received chemotherapy only. The sample size in each group was 31. The treatment outcomes of the patients were evaluated at the end of 2 months of treatment, including clinical manifestation, features of chest CT, conversion of acid-fast bacilli (AFB) smear examination and features under bronchoscope. The results were compared between two groups.  Results At the end of 2 months of treatment, the proportion of patients in the combined treatment group who had improvement of lesions under bronchoscope was 87.10% (27/31)， which was higher than that in the simple chemotherapy group (45.16%,14/31); there was a statistically significant difference between the two groups (χ²=12.17, P<0.01). The proportions of clinical manifestation improvement, AFB smear conversion and chest CT improvement among patients in the combined treatment group were all significantly higher than those in the control group (χ²=7.828，P<0.05; χ²=4.309, P<0.05 andχ²=3.865, P<0.05).  Conclusion The local infusion of anti-TB drugs by bronchoscopy combined with systemic chemotherapy is an effective method for treatment of bronchial TB, which can rapidly relieve patient’s symptoms, accelerate absorption of the lesion and make better overall treatment outcomes for the patients than simple chemotherapy.

Ratio of new smear positive tuberculosis notification and prevalence rate in different population and regions of China in 2010

XIA Yin-yin，CHENG Jun，ZHANG Hui，WANG Li-xia，DU Xin，CHEN Wei，LIU Xiao-qiu，LI Xue，JIANG Shi-wen，CHENG Shi-ming

Chinese Journal of Antituberculosis. 2014, 36(6):  498-502.  doi:10.3969/j.issn.1000-6621.2014.06.018

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Objective To analyze the case detection situation in different population and regions by calcula-ting ratio of smear positive tuberculosis notification rate and prevalence rate. Methods We used prevalence data obtained from the 5th national tuberculosis prevalence survey (52/100 000, 152/252 940) and notification rate obtained from national tuberculosis information management system (39/100 000 person-year, 428 214 000/1 088 190 000), calculated ratio of notification and prevalence rate by gender, age groups, regions and provinces whether implemented world bank funded tuberculosis control project or not, then did direct comparative analysis of them. Results In 2010, the ratio of new smear positive tuberculosis notification rate and prevalence was 0.75 per person-year (39/52) for people over 15 years old. This ratio is lower in male(0.72 per person-year, 55/76) than female (0.85 per person-year, 23/27), and varied in different age groups(every 10 years as a group), highest (1.55 per person-year, 31/20)among 15- group and lowest (0.43 per person-year, 79/185) among ≥75 years of age group. Central provin-ces had higher ratio of notification and prevalence rate (1.22 per person-year, 51/42) than eastern (0.95 per person-year, 36/37) and western provinces (0.57 per person-year, 49/85). Ratio of notification and prevalence rate in world bank funded tuberculosis control project provinces (0.90 per person-year, 48/53) was higher than non-project provinces (0.80 per person-year, 43/54). Conclusion Our research finds out that the ratio of notification and prevalence rate among elderly patients and in western provinces were relatively low which suggests that we should enhance case detection work among them. Provinces which had implemented world bank funded tuberculosis control project thus started using directly observed treatment short-course strategy longer has higher ratio of notification and prevalence rate which proves they get better performance on case detection.

Research progress on ESAT-6 family protein of Mycobacterium tuberculosis

YANG You-rong, WU Xue-qiong

Chinese Journal of Antituberculosis. 2014, 36(6):  503-508.  doi:10.3969/j.issn.1000-6621.2014.06.019

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At present, the pathogenesis of Mycobacterium tuberculosis (Mtb) and the defense mechanism of the host are not very clear. The study on the biological characteristics of Mtb protein antigen will contribute to understand these problems, and lay the foundation for the development of new tuberculosis vaccine, immune diagnostic reagents and drugs. The early secreted antigenic target 6 (ESAT-6) family proteins is a class of small molecular proteins with spiral structure, which exports the cell through the ESX secretion system. This family includes 23 members (EsxA-W), in which the genes coding two adjacent proteins in the genome form 11 gene pairs liking operon structure. These proteins involve in the interaction between the host and pathogen, are the immunodominant antigens in the recognition of human immune system, in which most of them are immunodominant T cell antigens, play a key role on the pathogenesis of Mtb and immune protection mechanism of the individual. There have been more reports on the EsxA and EsxB, but the researches on other members of the ESAT-6 family were less reported. Therefore, in this paper we briefly introduced the research progress of other ESAT-6 family members in order to lay the foundation for further research.

A functional analysis of the proteins targeted by prokaryotic ubiquitin-like protein (Pup) in mycobacteria

XUE Yu, LIU Yi, ZHANG Xu-xia, ZHANG Jun-jie, LI Chuan-you

Chinese Journal of Antituberculosis. 2014, 36(6):  509-513.  doi:10.3969/j.issn.1000-6621.2014.06.020

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Prokaryotic ubiquitin-like protein (Pup), a small protein alike to the ubiquitin in eukaryotes, exi-sts in mycobacterium. When targeted by the Pup, the proteins are degraded by the proteasome or become regulators after the modification. Thus, it can influence all activities of the cells. Currently, 58 population proteins, which can be targeted by Pup, have been identified in Mycobacterium tuberculosis (Mtb); in Mycobacterium smegmatis, it has been identified that 41 population proteins can be targeted by Pup, and the orthologous genes of 38 out of 41 population proteins can be found in Mtb genome. Those target proteins have been involved in at least 6 mechanisms and functions in the activities of the cells, including the virulence of mycobacterium, information pathway, lipid metabolism, cell wall and/or membrane synthesis, and intermediary metabolism, etc. After targeted, most of the population proteins are degrated by the proteasome and only a few of them are involved in the signal transmissions and re-gulations. Through comparisons and analysis of the target proteins, the further explanations can be made for the mechanisms of Mtb drug-resistance development and pathopoiesis; at the same time, the proteins targeted by Pup can be the potential drug targets, which provides the theory foundations for the TB treatment and control.