Boping Du

Prevalence and Risk Factors for Latent Tuberculosis Infection among Health Care Workers in China: A Cross-Sectional Study

Background Health care workers (HCWs) are at risk of latent tuberculosis infection (LTBI). In China, tuberculosis (TB) is a major public health problem, but the prevalence of LTBI in HCWs especially in the hospital for pulmonary diseases has not been assessed enough. The aim of this study was to determine the prevalence and putative risk factors of LTBI among HCWs in a chest hospital and a TB research institute in China. Methodology/Principal Findings A cross-sectional study was conducted among HCWs in China in 2012. LTBI was assessed by T-SPOT.TB, and information on HCWs was collected using a standardised questionnaire. Risk factors for LTBI were analyzed by univariate and multivariate regression. The overall prevalence of LTBI among HCWs was 33.6%. Analyzed by job category, the highest prevalence was found among laboratory staff (43.4%). In the different workplaces, the proportion of LTBI was significantly higher among the high risk workplaces (37.4%) compared to the low risk workplaces. The duration of employment had a significant impact on the prevalence of LTBI. Positive T-SPOT.TB test results accounted for 17.6%, 16.8%, 23.5%, 41.8% and 41.6% in groups of ≤2, 3–5, 6–10, 11–20, and >20 working years respectively. In multivariate analysis, job categories (Laboratory staff [2.76 (95% CI: 1.36; 5.60)], technician staff [2.02 (95% CI: 1.12; 3.64)]); working duration as a HCW for 11 to 20 years [3.57 (95% CI: 1.46; 8.71)], and 20 years above [3.41 (95% CI: 1.28; 9.11)]; and the history of household TB contact [2.47 (95% CI: 1.15; 5.33)] were associated with increased risk of LTBI. Conclusions/Significance Prevalence of LTBI estimated by T-SPOT.TB is high among Chinese HCWs and working duration, job category and the history of household TB contact were associated with increased risk. These data highlight adequate infection control measures should be undertaken.

Risk factors for false-negative T-SPOT.TB assay results in patients with pulmonary and extra-pulmonary TB

OBJECTIVES To investigate the risk factors for false-negative T-SPOT.TB results in patients with pulmonary TB (PTB) and extra-pulmonary TB (EPTB). METHODS Patients with suspected TB who underwent valid T-SPOT.TB tests were prospectively enrolled at Beijing Chest Hospital between November 2012 and November 2013. Basic characters and clinical laboratory findings were compared between true-positive and false-negative T-SPOT.TB groups. RESULTS Of 1928 suspected TB patients, 774 (530 PTB and 244 EPTB) microbiologically/histopathogenically-confirmed patients (636 culture-confirmed) were analyzed. Forty-six PTB patients (8.7%) and 32 EPTB patients (13.1%) had negative T-SPOT.TB results. Multivariate analysis showed that increased age [odds radio (OR) 2.26, 95% confidence interval (CI) 1.11-4.58], over-weight (BMI ≥ 25 kg/m(2), OR 2.43, 95% CI 1.05-5.63), and a longer period of illness before hospitalization (>6 months, OR 2.46, 95% CI 1.24-4.92) were independent risk factors for false-negative T-SPOT.TB results in PTB patients. In EPTB patients, increased age (OR 2.42, 95% CI 1.09-5.35) also showed an independent association with false-negative T-SPOT.TB results. CONCLUSION Careful interpretation of negative T-SPOT.TB results is necessary in older patients with suspected PTB or EPTB, and in PTB patients who are over-weight or have had longer periods of illness before hospitalization.

Cerebrospinal fluid metabolomic profiling in tuberculous and viral meningitis: Screening potential markers for differential diagnosis

BACKGROUND Tuberculous meningitis (TBM) is the most severe and frequent form of central nervous system tuberculosis. The current lack of efficient diagnostic tests makes it difficult to differentiate TBM from other common types of meningitis, especially viral meningitis (VM). Metabolomics is an important tool to identify disease-specific biomarkers. However, little metabolomic information is available on adult TBM. METHODS We used 1H nuclear magnetic resonance-based metabolomics to investigate the metabolic features of the CSF from 18 TBM and 20 VM patients. Principal component analysis and orthogonal signal correction-partial least squares-discriminant analysis (OSC-PLS-DA) were applied to analyze profiling data. Metabolites were identified using the Human Metabolome Database and pathway analysis was performed with MetaboAnalyst 3.0. RESULTS The OSC-PLS-DA model could distinguish TBM from VM with high reliability. A total of 25 key metabolites that contributed to their discrimination were identified, including some, such as betaine and cyclohexane, rarely reported before in TBM. Pathway analysis indicated that amino acid and energy metabolism was significantly different in the CSF of TBM compared with VM. CONCLUSIONS Twenty-five key metabolites identified in our study may be potential biomarkers for TBM differential diagnosis and are worthy of further investigation.

Evaluation of interferon-γ release assay in the diagnosis of osteoarticular tuberculosis.

The aim of this study was to assess the value of interferon-γ (IFN-γ) release assay (IGRA) (T-SPOT.TB) for patients with suspected osteoarticular tuberculosis (TB) in comparison with conventional and molecular methods. Of 145 patients with suspected osteoarticular TB, recruited from Beijing Chest Hospital between July 2011 and June 2012, 86 (59.3%)had osteoarticular TB (26 with culture-confirmed TB, 60 with probable TB), 24 (16.6%) were not having active TB. The remaining 17 (11.7%) inconclusive TB and 18 (12.4%) possible TB were excluded from final analysis. In addition to conventional tests and molecular method, T-SPOT.TB assay using peripheral blood mononuclear cells to examine IFN-γ response to early secretory antigenic target 6 and culture filtrate protein 10 was also performed. The sensitivity and specificity for T-SPOT.TB assay were 94.2% and 70.8%, respectively. A statistically significant difference in sensitivity was found between T-SPOT.TB assay (94.2%) and other tests (acid-fast bacilli smear (19.7%), culture (34.2%), real-time PCR (36.8%); P < 0.01, respectively). These results suggested that the IGRA assay could provide useful aids in the diagnosis of osteoarticular TB.

Diagnostic performance of interferon-γ release assay for lymph node tuberculosis

The aim of the study was to evaluate the performance of interferon-γ (IFN-γ) release assay (IGRA) (T-SPOT.TB) for patients with suspected lymph node tuberculosis (TB). Of the 405 patients with suspected lymph node TB, enrolled from Beijing Chest Hospital between July 2011 and April 2015, 83 (20.5%) were microbiologically/histopathologically confirmed lymph node TB, and 282 (69.6%) did not have active TB. The remaining 21 inconclusive TB and 19 clinical TB were excluded from the final analysis (9.9%). T-SPOT.TB using peripheral blood mononuclear cells was performed to examine the IFN-γ response to the Mycobacterium tuberculosis-specific antigens early secretory antigenic target 6 and culture filtrate protein 10. The overall sensitivity and specificity for T-SPOT.TB were 90.4% and 70.5%, respectively. Spot-forming cells in the lymph node TB group (184 [48-596/10(6) peripheral blood mononuclear cells {PBMCs}]) were significantly higher than that in the nonactive TB group (0 [0-41]/10(6) PBMCs) (P<0.001). These results suggest that the IGRA assay could be a useful aid in the diagnosis of lymph node TB.

Genome-wide transcriptional profiling identifies potential signatures in discriminating active tuberculosis from latent infection

To better understand the host immune response involved in the progression from latent tuberculosis infection (LTBI) to active tuberculosis (TB) and identify the potential signatures for discriminating TB from LTBI, we performed a genome-wide transcriptional profile of Mycobacterium tuberculosis (M.TB)–specific antigens-stimulated peripheral blood mononuclear cells (PBMCs) from patients with TB, LTBI individuals and healthy controls (HCs). A total of 209 and 234 differentially expressed genes were detected in TB vs. LTBI and TB vs. HCs, respectively. Nineteen differentially expressed genes with top fold change between TB and the other 2 groups were validated using quantitative real-time PCR (qPCR), and showed 94.7% consistent expression pattern with microarray test. Six genes were selected for further validation in an independent sample set of 230 samples. Expression of the resistin (RETN) and kallikrein 1 (KLK1) genes showed the greatest difference between the TB and LTBI or HC groups (P < 0.0001). Receiver operating characteristic curve (ROC) analysis showed that the areas under the curve (AUC) for RETN and KLK1 were 0.844 (0.783–0.904) and 0.833 (0.769–0.897), respectively, when discriminating TB from LTBI. The combination of these two genes achieved the best discriminative capacity [AUC = 0.916 (0.872–0.961)], with a sensitivity of 71.2% (58.7%–81.7%) and a specificity of 93.6% (85.7%–97.9%). Our results provide a new potentially diagnostic signature for discriminating TB and LTBI and have important implications for better understanding the pathogenesis involved in the transition from latent infection to TB activation.

Diagnosis of latent tuberculosis by ELISPOT assay and tuberculin skin test.

OBJECTIVE To determine the prevalence of latent tuberculosis infection (LTBI) in college students. PATIENTS AND METHODS Four hundred and twenty newly admitted college students were enrolled. The Enzyme-Linked ImmunoSpot assay (ELISPOT) was used. Overall, 171 students with ELISPOT assay+/TST+ were monitored for three years to detect active TB disease. RESULTS The overall positive rate of ELISPOT assay was 40.7% among TST+ students. The ELISPOT positive rates were 36.9%, 45.4%, and 64.3% in groups of TST induration of 10-14mm, 15-20mm, and ≥20mm, respectively, with a significant difference (χ(2)=10.136, P<0.01) but no significant difference between BCG scar and no scar (41.2% vs. 38.8%; P>0.05). None of the 171 untreated students contracted active TB within the three-year monitoring period. CONCLUSION The LTBI rate might be overestimated by TST compared with interferon-γ release assays. On the basis of a close monitoring, few students developed active TB despite a positive result to the TST and ELISPOT assay.

Prospective Comparison of QFT-GIT and T-SPOT.TB Assays for Diagnosis of Active Tuberculosis

T-SPOT.TB and QuantiFERON-TB Gold In-Tube (QFT-GIT) tests, as two commercial blood assays for diagnosing active tuberculosis (ATB), are not yet fully validated. Especially, there are no reports on comparing the efficacy between the two tests in the same population in China. A multicenter, prospective comparison study was undertaken at four hospitals specializing in pulmonary diseases. A total of 746 suspected pulmonary TB were enrolled and categorized, including 185 confirmed TB, 298 probable TB and 263 non-TB. Of 32 patients with indeterminate test results (ITRs), age and underlying disease were associated with the rate of ITRs. Furthermore, the rate of ITRs determined by T-SPOT.TB was lower than QFT-GIT (0.4% vs. 4.3%, P < 0.01). When excluding ITRs, the sensitivities of T-SPOT.TB and QFT-GIT were 85.2% and 84.8%, and specificities of 63.4% and 60.5%, respectively in the diagnosis of ATB. The two assays have an overall agreement of 92.3%, but exhibited a poor linear correlation (r2 = 0.086) between the levels of interferon-γ release detected by the different assays. Although having some heterogeneity in detecting interferon-γ release, both the QFT-GIT and T-SPOT.TB demonstrated high concordance in diagnosing ATB. However, neither of them showed suitability in the definitive diagnosis of the disease.

MiRNA expression profiles of serum exosomes derived from individuals with latent and active tuberculosis

Tuberculosis (TB) has become a leading cause of death worldwide, which is largely attributed to the difficulties in diagnosis and treatment of TB patients. Exosomes carrying RNA, particularly miRNA, have been indicated their functional and diagnostic potential in diseases, including tuberculosis (TB). In the present study, we performed RNA-seq based analysis on exosomal miRNA profiles for clinical specimens of healthy controls (HC), active tuberculosis (TB) and latent tuberculosis infection (LTBI). We identified many distinct up-regulated and down-regulated differentially expressed miRNA and further screened top 20 in each compared groups which might provide a potential panel for differentiation of HC, LTBI, and TB. We classified all the differentially expressed miRNAs into six expression patterns and identified three persistently up-regulated miRNA (hsa-miR-140-3p, hsa-miR-3184-5p and hsa-miR-423-3p) as potential markers during TB progression. Combined with our previously detected exsomal mRNA, we screened the genes overlapped with predicted mRNA targets of differentially expressed miRNA and analyzed their involvement in Biological Process, indicating a decreased signaling transduction and increased cell death in LTBI and TB. Our results indicate the selective packaging of RNA cargoes into exosomes under different stages of Mycobacterium tuberculosis infection and facilitate further study of TB pathogenesis and development. IMPORTANCE The main reason for failure to eliminate TB is lack of understanding molecular mechanism of TB pathogenesis and difficulties in TB diagnosis and treatment. Exosomes provide a promising research tool because they are released from various cells containing valuable biochemical information related to diseases. We reveale distinct miRNA expression profile of the exosomes, which indicates selective packaging of RNA cargoes into exosomes under different stages of Mycobacterium tuberculosis infection. Further, we also provide evidence of related miRNA candidates potentially involving in TB progression and facilitating discovery of TB biomarkers.

Label-Free Quantitative Proteomics Identifies Novel Plasma Biomarkers for Distinguishing Pulmonary Tuberculosis and Latent Infection

The lack of effective differential diagnostic methods for active tuberculosis (TB) and latent infection (LTBI) is still an obstacle for TB control. Furthermore, the molecular mechanism behind the progression from LTBI to active TB has been not elucidated. Therefore, we performed label-free quantitative proteomics to identify plasma biomarkers for discriminating pulmonary TB (PTB) from LTBI. A total of 31 overlapping proteins with significant difference in expression level were identified in PTB patients (n = 15), compared with LTBI individuals (n = 15) and healthy controls (HCs, n = 15). Eight differentially expressed proteins were verified using western blot analysis, which was 100% consistent with the proteomics results. Statistically significant differences of six proteins were further validated in the PTB group compared with the LTBI and HC groups in the training set (n = 240), using ELISA. Classification and regression tree (CART) analysis was employed to determine the ideal protein combination for discriminating PTB from LTBI and HC. A diagnostic model consisting of alpha-1-antichymotrypsin (ACT), alpha-1-acid glycoprotein 1 (AGP1), and E-cadherin (CDH1) was established and presented a sensitivity of 81.2% (69/85) and a specificity of 95.2% (80/84) in discriminating PTB from LTBI, and a sensitivity of 81.2% (69/85) and a specificity of 90.1% (64/81) in discriminating PTB from HCs. Additional validation was performed by evaluating the diagnostic model in blind testing set (n = 113), which yielded a sensitivity of 75.0% (21/28) and specificity of 96.1% (25/26) in PTB vs. LTBI, 75.0% (21/28) and 92.3% (24/26) in PTB vs. HCs, and 75.0% (21/28) and 81.8% (27/33) in PTB vs. lung cancer (LC), respectively. This study obtained the plasma proteomic profiles of different M.TB infection statuses, which contribute to a better understanding of the pathogenesis involved in the transition from latent infection to TB activation and provide new potential diagnostic biomarkers for distinguishing PTB and LTBI.