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

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 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

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

Polymerase chain reaction (PCR) using primers targeting the IS6110 repetitive sequence was employed to detect Mycobacterium tuberculosis in 228 samples from patients with tuberculosis or other pulmonary diseases and controls, and the results were compared with culture and clinical findings. None of culture negative samples from 17 healthy controls were PCR positive. Of 109 active tuberculosis patients under chemotherapy, 88 (80.7%) were PCR positive and were significantly higher than 63 (57.8%) positive by culture. Fifty-nine (93.7) of 63 culture positive and 29 (63.0%) of 46 culture negative specimens contained M. tuberculosis detectable by PCR. In 41 specimens from inactive tuberculosis patients who visited to the chest clinic because of chest problems, 16 (39.0%) also gave PCR positive results. In addition, 14 (46.7%) of 30 specimens submitted for M. tuberculosis culture from patients with pulmonary diseases were PCR positive. Presumptive diagnosis of these PCR positive patients was bronchitis, pneumonia, bronchial asthma, etc. Therefore, this study suggests that PCR is sensitive and specific in detecting M. tuberculosis in clinical specimens. However, the interpretation of the PCR results in specimens from patients with pulmonary diseases should be done cautiously in areas with a high prevalence of tuberculosis.
Base Sequence ; DNA, Bacterial/analysis ; Human ; Lung Diseases/*microbiology ; Molecular Sequence Data ; Mycobacterium tuberculosis/genetics/*isolation & purification ; *Polymerase Chain Reaction ; Sputum/microbiology ; Support, Non-U.S. Gov't ; Tuberculosis, Pulmonary/diagnosis/*microbiology

Abscess ; diagnosis ; etiology ; therapy ; Antitubercular Agents ; therapeutic use ; DNA, Bacterial ; analysis ; Debridement ; methods ; Diagnosis, Differential ; Follow-Up Studies ; Humans ; Male ; Metacarpus ; Middle Aged ; Mycobacterium tuberculosis ; genetics ; isolation & purification ; Tuberculosis, Pulmonary ; complications ; drug therapy ; microbiology

We evaluate the performance of Xpert MTB/RIF for the diagnosis of extrapulmonary tuberculosis (EPTB) in China. The performance of Xpert was evaluated compared to the composite reference standard (CRS), drug susceptibility testing (DST), and imaging examination. The overall sensitivity and specificity of Xpert were 64.1% (195/304) and 100% (24/24), respectively, using CRS as the gold standard. The sensitivity was significantly higher than that of culture for pus (P<0.05). The proportion of EPTB-positive cases diagnosed by imaging was two times more than that diagnosed using Xpert; however, 6 out of 19 cases may have been overdiagnosed by imaging. Compared to phenotypic DST, the sensitivity and specificity of Xpert were 80% (12/15) and 100% (75/75), respectively. Considering its high sensitivity and specificity, Xpert MTB/RIF may be used as a rapid initial test for EPTB diagnosis, and may also support a quicker decision on the treatment regimen. The combination of imaging and Xpert testing could provide high efficiency and accurate diagnosis of suspected EPTB.
Bacterial Proteins ; genetics ; metabolism ; China ; DNA-Directed RNA Polymerases ; genetics ; metabolism ; Diagnostic Tests, Routine ; instrumentation ; methods ; Drug Resistance, Bacterial ; Humans ; Microbial Sensitivity Tests ; Mycobacterium tuberculosis ; drug effects ; genetics ; isolation & purification ; metabolism ; Retrospective Studies ; Rifampin ; pharmacology ; Sensitivity and Specificity ; Sputum ; Tuberculosis ; Tuberculosis, Pulmonary ; diagnosis ; microbiology