OBJECTIVETo explore the association between the genetic polymorphisms of mannose-binding protein (MBP) alleles and susceptibility to pulmonary tuberculosis.

METHODS125 pulmonary tuberculosis cases and 198 healthy controls were collected. A case-control study was conducted. Three structural gene mutations in exon 1 of MBP gene (codon 52, codon 54 and codon 57) were studied. Polymerase chain reaction with sequence-specific primers (PCR-SSP) was carried out in the polymorphism in MBP alleles. Information on related risk factors of tuberculosis was collected, using a pre-tested questionnaire. Univariate and multivariate logistic analyses were conducted with SPSS software package.

RESULTSThe frequencies of mutant heterozygote or homozygote of MBP-52, 54, 57 were 8.0%, 7.2% and 0.4% for cases and 5.3%, 4.3%, 0.5% for controls, respectively. The distribution of mutant genotypes of MBP did not show significant difference between tuberculosis patients and control by Mantel-Haenszel chi2 on sex. The univariate analysis demonstrated that body mass index, marital status, vaccinal vestige, bacillus of Calmette-Guerin vaccine immunization, contacted with pulmonary tuberculosis patients, familial traits were the risk factors of pulmonary tuberculosis. After adjusting those related environmental factors in the multivariate logistic analyses, the total MBP (MBP-52, MBP-54 and MBP-57) and MBP-52 heterozygote genotypes were significantly overrepresented in cases, with adjusted OR (95% CI) being 2.182 (1.058-4.499) and 2.574 (1.028-6.446).

CONCLUSIONTotal MBP and MBP-52 mutant genotypes might be associated with the susceptibility to pulmonary tuberculosis.

Case-Control Studies ; Genetic Predisposition to Disease ; Humans ; Mannose-Binding Lectin ; genetics ; Polymorphism, Genetic ; Tuberculosis, Pulmonary ; genetics

Mycobacterium lentiflavum has recently been described as an emerging human pathogen without regard to the immune status of the host. We herein report on M. lentiflavum isolated from a respiratory specimen of a patient. Although the organism described in this case seems to be a colonizer of a lung destroyed by tuberculosis, the current methods for species identification of nontuberculous mycobacteria have to be re-evaluated so as not to underestimate these organisms.
Aged ; Humans ; Male ; Mycobacterium/genetics/*isolation & purification ; Tomography, X-Ray Computed ; Tuberculosis, Pulmonary/diagnosis/*microbiology/radiography

The AdvanSure tuberculosis/non-tuberculous mycobacterium (TB/NTM) PCR (LG Life Science, Korea) and COBAS TaqMan Mycobacterium tuberculosis (MTB) PCR (Roche Diagnostics, USA) are commonly used in clinical microbiology laboratories. We aimed to evaluate these two commercial real-time PCR assays for detection of MTB in a large set of clinical samples over a two-year period. AdvanSure TB/NTM PCR and COBAS TaqMan MTB PCR were performed on 9,119 (75.2%) and 3,010 (24.8%) of 12,129 (9,728 respiratory and 2,401 non-respiratory) MTB specimens, with 361 (4.0%) and 102 (3.4%) acid-fast bacilli (AFB)-positive results, respectively. In MTB culture, 788 (6.5%) MTB and 514 (4.2%) NTM were identified. The total sensitivity and specificity of the AdvanSure assay were 67.8% (95% confidence interval [CI], 63.9-71.6) and 98.3% (95% CI, 98.0-98.6), while those of the COBAS TaqMan assay were 67.2% (95% CI, 60.0-73.8) and 98.4% (95% CI, 97.9-98.9), respectively. The sensitivities and specificities of the AdvanSure and COBAS TaqMan assays for AFB-positive and AFB-negative samples were comparable. Furthermore, the AdvanSure assay showed fewer invalid results compared with the COBAS TaqMan assay (5.0 vs. 20.4 invalid results/1,000 tests, P<0.001). AdvanSure assay represents a comparable yet more reliable method than COBAS TaqMan for the identification of mycobacteria in routine clinical microbiology.
DNA, Bacterial/genetics/metabolism ; Humans ; Mycobacterium tuberculosis/*genetics/isolation & purification ; Reagent Kits, Diagnostic ; Real-Time Polymerase Chain Reaction ; Republic of Korea ; Sensitivity and Specificity ; Tuberculosis, Pulmonary/diagnosis

BACKGROUND: Although there are many methods including AFB smear and culture, and the analysis of pleural fluid in the etiological diagnosis of pleural effusion, it is sometimes difficult to confirm a diagnosis especially in cases of incomplete pleural biopsies. Moreover, the high incidence of tuberculous pleuritis in young people caused confusion in the differential diagnosis of pleural effusion in Korea. The pathognomonic finding of tuberculous pleuritis in pleural biopsy is chronic granulomatous pleuritis (CGP) with caseous necrosis. But a biopsy does not always provide a definitive diagnosis, which shows in only 60~70% of all biopsies, because of either limitations in blind biopsies or inadequate specimens. An adequate biopsy also gives only limited information, such as chronic or nonspecific pleuritis. METHODS: We compared the clinical diagnosis, pathologic findings and detection of mycobacterial DNA using nested PCR of pleural biopsy tissues. We carried out the nested PCR for IS6110 insertion sequence of Mycobacterium tuberculosis using outer primer IS-1/IS-2 (5'-AGGCGTTGGTTCGCGAGGG-3' /5'-TGATGACGCCCTCGTTGCC-3') and inner primer IS-3/IS-4 (5'-CCAACCCGCTCGGTCTCAA-3' /5'-ACCGATGGACTGGTCACCC-3') in 52 pleural biopsy tissues which were pathologically diagnosed as tuberculous pleuritis, malignant pleuritis or non-specific pleuritis. RESULTS: Five (71.4%) of 7 cases clinically and pathologically confirmed tuberculous pleuritis diagnosed as chronic granulomatous pleuritis (CGP) with caseous necrosis revealed positive in nested PCR for M. tuberculosis. Seven (36.8%) of 19 cases diagnosed as CGP without caseous necrosis were positive. However, only 3 (25%) of 12 cases diagnosed as non-specific chronic pleuritis were positive by PCR for M. tuberculosis. Neither congestive heart failure nor malignancies with pleurisy showed a positive reaction. CONCLUSION: In this study, pathologic findings were significantly associated with the detection rate of mycobacterial DNA. And, even in patients with nonspecific or chronic inflammatory pleuritis, mycobacterial DNA could be detected by using nested PCR in pleural biopsy tissue with good specificity. Detection of mycobacterial DNA in pleural tissue might provide additional information for etiological diagnosis in patients with pleural effusion.
Biopsy ; Comparative Study ; DNA, Bacterial/*isolation & purification ; Human ; Lung/microbiology/*pathology ; Mycobacterium tuberculosis/*genetics/isolation & purification ; *Polymerase Chain Reaction ; Support, Non-U.S. Gov't ; Tuberculosis, Pulmonary/*diagnosis/genetics/microbiology

OBJECTIVETo establish a recombinant plasmid of CFP32 of Mycobacterium tuberculosis in E. coli, and to analyze its antigenicity.

METHODSRv0577 gene was amplified by polymerase chain reaction from genome of Mycobacterium tuberculosis, and then cloned into vector pMD18-T followed by the subclone into the expression vector pET21a. Recombinant CFP32 was expressed and purified. The antigenicity of the recombinant protein was analyzed by using Western-blot. The purified recombinant CFP32 protein was used as an antigen to screen the sera of 7 pulmonary TB patients (n = 97), as well as the other pulmonary disease patients (n = 25), and the clinically healthy controls (n = 38) by ELISA.

RESULTSRecombinant plasmid of CFP32 was established, and be expressed efficiently in E. coli BL21 (DE3). The relative molecular mass of the protein was about 300,000 by SDS-PAGE analysis. The protein purified by Ni-NTA was in a purity over 90%, which was confirmed by Western-blot analysis. ELISA analysis showed its sensitivity and specificity were 63.9% (62/97) and 96.8% (2/63) respectively.

CONCLUSIONThe recombinant expression plasmid pET21a CFP32 has been constructed and CFP32 proteins has been successfully expressed and be purified in E. coli and, ELISA analysis has identified the recombinant CFP32 as a candidate antigen for TB serodiagnosis.

Antigens, Bacterial ; blood ; Bacterial Proteins ; genetics ; immunology ; Cloning, Molecular ; Escherichia coli ; Gene Expression ; Humans ; Mycobacterium tuberculosis ; genetics ; isolation & purification ; Plasmids ; Recombinant Proteins ; Serologic Tests ; Tuberculosis, Pulmonary ; diagnosis ; microbiology

BACKGROUNDEarly detection of pulmonary tuberculosis (PTB) is a big challenge in smear negative and sputum scarce patients in China. Simultaneous amplification and testing methods for detection of the Mycobacterium tuberculosis (MTB) complex (SAT-TB assay) is a novel molecular technique established in our hospital. This method has a high sensitivity and specificity in the lab. In this study, the clinical diagnostic performance of this method in smear-negative or sputum-scarce PTB suspects was investigated and evaluated.

METHODSTwo hundred smear negative and 80 sputum-scarce patients were recruited in this study. Samples that included sputum or bronchial washing fluid were collected and sent for both bacteria culture and SAT-TB assay. Diagnosis for these patients was based on the comprehensive evaluation of chestX- ray/CT study, histology examination, lab results, and treatment response. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for each diagnostic test were investigated and calculated using confirmed tuberculosis (TB) and non-TB cases. The time required for detection of MTB was also measured for each method.

RESULTSNinety-two patients (33%) were diagnosed as definitive TB, 112 patients (40%) were probable PTB, and 76 (27%) were non-TB. The sensitivity, specificity, PPV, and NPV of SAT-TB in smear-negative PTB suspects were 93% (95% CI, 84%-98%), 98% (95% CI, 90%-100%), 98% (95% CI, 91%-100%), and 93% (95% CI, 83%-98%). In sputum scarce PTB suspects, the sensitivity, specificity, PPV, and NPV of the SAT-TB assay on bronchial washing fluids were 90% (95% CI, 74%-98%), 100% (95% CI, 85%-100%), 100% (95% CI, 88%-100%), and 88% (95% CI, 69%-97%). The accuracy of the SAT-TB assay is consistent with the bacteria culture assay. The median time required for detecting MTB in the SAT-TB assay was 0.5 day, which was much faster than bacteria culture (28 days).

CONCLUSIONSThe SAT-TB assay is a fast and accurate method for the detection of MTB. It can be widely applied in the clinic and be an asset in early detection and management of PTB suspects, especially in those patients who are smear negative or sputum scarce.

Adult ; China ; Female ; Humans ; Male ; Middle Aged ; Mycobacterium tuberculosis ; genetics ; pathogenicity ; Nucleic Acid Amplification Techniques ; methods ; Sputum ; microbiology ; Tuberculosis, Pulmonary ; diagnosis ; Young Adult

OBJECTIVETo study the characteristics of drug-resistant genetic mutation of rpoB in multiple drugs resistant bacillus tuberculosis (MDR-TB) among patients of pneumoconiosis complicated with pulmonary tuberculosis.

METHODSA total of 114 clinical isolated strains of Mycobacterium tuberculosis were collected, MDR-TB were identified by conventional antimicrobial susceptibility test (AST). Their genomes DNA were extracted, the target genes were amplified by PCR, and the hot regions in the rpoB gene were analyzed by automated DNA sequenator.

RESULTSThe results by AST showed that there were 31 strains of MDR-TB in the 114 clinical isolated strains, the rate of drug resistance was 27.19% (31/114). No mutation of rpoB was identified in 10 rifampicin-sensitive strains that randomly chosen, while conformation changes were found in MDR-TB strains, and the mutation rate of rpoB was 93.55% (29/31) in resistant strains, mainly concentrated in codon 531 (45.16%, 14/31) and 526 (29.03%, 9/31), happened base substitutions, including 27 unit point mutation and 2 two point mutation.

CONCLUSIONSThe substitution of highly conserved amino acids encoded by rpoB gene results in the molecular mechanism responsible for RFP resistance in MDR-TB among patients of pneumoconiosis complicated with pulmonary tuberculosis. It also proves that rpoB gene is diversiform.

Adult ; Aged ; Bacterial Proteins ; genetics ; DNA-Directed RNA Polymerases ; Drug Resistance, Multiple, Bacterial ; genetics ; Genes, Bacterial ; Humans ; Male ; Microbial Sensitivity Tests ; Middle Aged ; Mining ; Mutation Rate ; Mycobacterium tuberculosis ; drug effects ; genetics ; isolation & purification ; Pneumoconiosis ; microbiology ; Sequence Analysis ; Tuberculosis, Pulmonary ; microbiology

OBJECTIVETo investigate the feasibility of real-time fluorescent quantitative (qPCR) assay in detecting mycobacterium tuberculosis complex (MTB) in paraffin embedded tissues for diagnostic purpose.

METHODSUsing qPCR assay, 1000 consecutive formalin-fixed and paraffin embedded (FFPE) tissues (from 2011 to 2012) suspected of MTB infection were tested by amplifying the MTB specific insertion sequence 6110 (IS6110). The specificity of the PCR product was confirmed by Sanger sequencing as compared with the MTB genomic DNA of the IS6110 sequence. Tissues with Ziehl-Neelsen acid-fast staining were used as control.

RESULTSIn the 1000 samples, 513 were positive for mycobacterium by Ziehl-Neelsen acid-fast staining (detection rate 51.3%); whereas 546 were MTB positive by qPCR assay (detection rate 54.6%). Concordance rate for both assays was 73.1%. The diagnosis rate increased by 14.4% by combinination of Ziehl-Neelsen acid-fast staining and qPCR results. More interestingly, by analyzing the Ziehl-Neelsen acid-fast staining and qPCR results three cases of M.leprae infection and four cases of non-tuberculous Mycobacterium (NTM) infection were identified.

CONCLUSIONSqPCR detection of MTB in FFPE tissue is more sensitive than Ziehl-Neelsen acid-fast staining assay. Combination of these two assays can increase the detection rate and also identify some rare cases of NTM infection.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child ; DNA, Bacterial ; genetics ; Female ; Humans ; Male ; Middle Aged ; Mycobacterium tuberculosis ; genetics ; isolation & purification ; Paraffin Embedding ; Real-Time Polymerase Chain Reaction ; Sequence Analysis, DNA ; Staining and Labeling ; methods ; Tuberculosis ; diagnosis ; microbiology ; Tuberculosis, Gastrointestinal ; diagnosis ; microbiology ; Tuberculosis, Lymph Node ; diagnosis ; microbiology ; Tuberculosis, Pulmonary ; diagnosis ; microbiology ; Young Adult

A case-control study was carried out that involved 203 individuals diagnosed with pulmonary tuberculosis (PTB) and 203 healthy subjects. Genotyping of TLR1 rs5743551 and rs5743618 polymorphisms was done using polymerase chain reaction-restriction fragments length polymorphism assay. We found that TLR1 rs5743551 variant affected the risk of PTB in the codominant (OR=3.28, 95% CI=1.98-5.45, P<0.0001, GA vs. GG; OR=1.86, 95% CI=1.05-3.28, P=0.033, AA vs. GG) and dominant (OR=2.69, 95% CI=1.67-4.34, P<0.0001, GA+AA vs. GG) inheritance models tested. The A allele was associated with a higher risk of PTB than the G allele (OR=1.33, 95% CI=1.01-1.75, P=0.049). The TG genotype of the rs5743618 variant significantly increased the risk of PTB compared to the risk associated with the TT genotype (OR=3.29, 95% CI=1.82-5.97, P<0.0001). The G allele was associated with a higher risk of PTB than the T allele (OR=3.00, 95% CI=1.69-5.31, P=0.0001). Our findings revealed that TLR1 rs5743551 and rs5743618 polymorphisms affected the risk of PTB in an Iranian population sample. Additional studies with larger sample sizes and involving subjects of different ethnicities are required to validate our present findings.
Adult ; Case-Control Studies ; Female ; Humans ; Iran ; epidemiology ; Male ; Middle Aged ; Polymorphism, Genetic ; Risk Factors ; Toll-Like Receptor 1 ; genetics ; Tuberculosis, Pulmonary ; epidemiology ; genetics

OBJECTIVEThe effect of rs4331426 polymorphism in the Chr18q11.2 locus on pulmonary tuberculosis (PTB) risk was evaluated.

METHODSThis case-control study included 208 PTB patients and 204 healthy subjects. Genotyping of the rs4331426 variant was conducted using polymerase chain reaction restriction fragment length polymorphism.

RESULTSThe frequencies of genotypes AA, AG, and GG polymorphisms were 83.1%, 15.9%, and 1.0% in the PTB group and 84.3%, 15.2%, and 0.5% in the control group, respectively. The frequency of the minor (G) allele was 8.9% in the PTB group and 8.1% in controls. Neither genotype nor allele frequencies of the rs4331426 variant showed statistically significant differences between PTB and controls. In addition, stratification by sex showed no significant association between the rs4331426 variant and PTB risk in males or females.

CONCLUSIONIn conclusion, the results of this study do not support an association between the rs4331426 polymorphism and risk of PTB in an Iranian population.

Adult ; Aged ; Case-Control Studies ; Chromosomes, Human, Pair 18 ; genetics ; Female ; Genetic Predisposition to Disease ; Humans ; Iran ; epidemiology ; Male ; Middle Aged ; Polymorphism, Genetic ; Tuberculosis, Pulmonary ; epidemiology ; genetics