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

BACKGROUND: The radiographic characteristics of tuberculous pneumonia in adults are similar to primary tuberculosis that occurs in childhood, and upper lobe cavitary tuberculosis is the hallmark of postprimary tuberculosis. The purpose of this study was to investigate the factors associated with tuberculous pneumonia by making comparison with cavitary tuberculosis. METHODS: The medical records and radiographic findings of patients with cavitary tuberculosis and tuberculous pneumonia, and who were diagnosed between March 2003 and February 2006, were analyzed retrospectively. RESULTS: Forty patients had cavitary tuberculosis and sixteen patients had tuberculous pneumonia. Fever was more frequent for tuberculous pneumonia, whereas hemoptysis was more frequent for cavitary tuberculosis. The duration of symptoms before visiting the hospital was shorter, but the diagnosis after admission was more delayed for tuberculous pneumonia patients than for cavitary tuberculosis patients. The prevalence of underlying comorbidities such cancer, diabetes, alcoholism and long-term steroid use was not different between the two groups. The patients with tuberculous pneumonia were older and they had lower levels of serum albumin and hemoglobin than those with cavitary tuberculosis. The patients with tuberculous pneumonia showed a tendency to have more frequent endobronchial lesion. Tuberculous pneumonia occurred in any lobe, whereas the majority of cavitary tuberculosis patients had upper lung lesion, but the prevalence of lymphadenopathy, pleural effusion and previous tuberculosis scar was not different between the two groups. CONCLUSIONS: Older age, a lower level of serum albumin and hemoglobin and a random distribution of lesion were associated with tuberculosis pneumonia as compared with cavitary tuberculosis. These findings suggest that the pathogenesis of tuberculous pneumonia might be different from that of cavitary tuberculosis.
Tuberculosis, Pulmonary/blood/*diagnosis/microbiology ; Tomography, X-Ray Computed ; Sputum/microbiology ; Severity of Illness Index ; Serum Albumin/metabolism ; Retrospective Studies ; Radiography, Thoracic ; Pneumonia, Bacterial/blood/*diagnosis/microbiology ; Mycobacterium tuberculosis/isolation & purification ; Middle Aged ; Male ; Lung/microbiology/pathology/radiography ; Humans ; Hemoglobins/metabolism ; Female ; Diagnosis, Differential ; Bronchoscopy ; Biopsy ; Adult

OBJECTIVETo study the values of a combination of multiple less invasive or non-invasive examinations including chest computed tomography (CT) scan, purified protein derivative (PPD) test, erythrocyte sedimentation rate (ESR) test, and C-reactive protein (CRP) test in the diagnosis of pediatric sputum-negative pulmonary tuberculosis (TB).

METHODSA retrospective analysis was performed on the clinical data of 269 children with confirmed pulmonary TB. Clinical symptoms and test results were analyzed and compared between the sputum-negative group (161 patients) and the sputum-positive group (108 patients).

RESULTSThe sputum-negative group had atypical clinical symptoms, with fewer typical or relatively specific imaging features compared with the sputum-positive group. The positive rates of PPD, ESR, and CRP tests for the sputum-negative group were 39.1%, 44.1%, and 56.5%, respectively, versus 55.6%, 79.6%, and 59.3% for the sputum-positive group. There were significant differences in the positive rates of PPD and ESR tests between the two groups (P<0.05). More than 80% of the patients in each group were diagnosed with pulmonary TB according to three or four less invasive or non-invasive tests, without significant difference in the positive rate between the two groups (P>0.05). Forty-six patients in the sputum-negative group underwent bronchoscopy, and morphological changes with a diagnostic value and/or etiological and pathological evidence were observed in 40 (87.0%) of them.

CONCLUSIONSThe diagnosis rate of pediatric sputum-negative pulmonary TB can be increased by combining tests including chest CT scan, PPD test, ESR test, and CRP test. Bronchoscopy is a reliable method for the auxiliary diagnosis of pediatric sputum-negative pulmonary TB if the combining tests cannot provide compelling evidence.

Adolescent ; Blood Sedimentation ; Bronchoscopy ; C-Reactive Protein ; analysis ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Sputum ; microbiology ; Tomography, X-Ray Computed ; Tuberculin Test ; Tuberculosis, Pulmonary ; diagnosis

PURPOSE: We investigated the value of an interferon-gamma release assay (IGRA) for the diagnosis of active pulmonary tuberculosis (PTB) among sputum smear negative PTB suspects in an environment with intermediate burden of PTB and high Bacillus Calmette-Guerin (BCG) vaccination rate. MATERIALS AND METHODS: We retrospectively reviewed IGRA, medical records, chest PA and CT scan of PTB suspects seen at Gangnam Severance Hospital, Seoul, Korea from Oct. 2007 to Apr. 2013. "Active PTB" was diagnosed when 1) M. tuberculosis culture positive, 2) confirmation by pathologic examination; or 3) clinical findings compatible with TB. RESULTS: Of 224 sputum smear negative PTB suspects, 94 were confirmed as having active PTB. There were no statistically significant differences in the diagnostic yield of IGRA between immunocompromised and immunocompetent sputum smear negative PTB suspects. IGRA did show superior sensitivity [81.9%, 95% confidence interval (CI); 74.13-89.70%] in the diagnosis of sputum smear negative PTB when compared with chest high-resolution computed tomography (HRCT), tuberculin skin test (TST), and chest X-ray (p<0.001). Also, IGRA showed highest negative predictive value (82.7%, 95% CI; 75.16-90.15%) when compared with HRCT, TST and chest X-ray (p=0.023). However, combining the results of IGRA with those of HRCT, TST, or both did not increase any diagnostic parameters. CONCLUSION: Failure to increase diagnostic yields by combination with other diagnostic modalities suggests that additional enforcement with IGRA may be insufficient to exclude other diagnoses in sputum smear negative PTB suspects and to screen active PTB in an environment with intermediate TB prevalence and a high BCG vaccination rate.
Adult ; Aged ; Female ; Humans ; Interferon-gamma Release Tests/*methods ; Male ; Middle Aged ; Retrospective Studies ; Sputum/*microbiology ; Tuberculosis, Pulmonary/blood/*diagnosis/metabolism