[Comparison of interferon-gamma release assays and adenosine deaminase of pleural fluid for the diagnosis of pleural tuberculosis].

OBJECTIVE: To compare the diagnostic performance of interferon gamma releasing assays (T-SPOT. TB) and adenosine deaminase (ADA) in pleural tuberculosis, and therefore to evaluate the value of T-SPOT. TB in a high tuberculosis burden country. METHODS: From June 2011 to November 2012, 111 patients with pleural fluid in Beijing Chest Hospital, Capital Medical University were enrolled prospectively and categorized as culture/biopsy-confirmed pleural tuberculosis group (n = 59) and non-pleural tuberculosis group (n = 52). Patients with uncertain diagnosis and clinically diagnosed pleural tuberculosis were excluded from the study. Pleural fluid T-SPOT. TB and ADA measurements were performed, in addition to other routine laboratory tests. Continuous variables (spot forming cells, SFCs) were compared using nonparametric Mann-Whitney U test. Comparisons between proportions were performed using Chi-squared test. RESULTS: The receiver operating characteristic (ROC) curve and cut-off value of pleural fluid T-SPOT. TB were established according to spot forming cells (SFC) between culture/biopsy-confirmed pleural tuberculosis group and non-pleural tuberculosis group (216 SFC/10(6) pleural fluid mononuclear cells). The sensitivity of pleural fluid T-SPOT. TB and ADA was 91.5% (54/59) and 71.2% (42/59), respectively. The specificity was 90.4% (47/52) and 92.0% (46/50), respectively. The sensitivity of pleural fluid T-SPOT. TB was significantly higher than that of ADA (χ(2) = 8.045, P < 0.01). There was no significant difference of specificity between pleural fluid T-SPOT. TB and ADA (χ(2) = 0.000, P > 0.05). The area under the ROC curve was 0.912 for pleural fluid T-SPOT. TB and 0.903 for ADA. The sensitivity of combination diagnosis of ADA and pleural fluid T-SPOT. TB decreased to 67.8% (40/59), but the specificity increased to 100.0% (50/50). CONCLUSIONS: Pleural fluid T-SPOT. TB are relatively accurate supplementary assays for the diagnosis of pleural tuberculosis in this high tuberculosis burden country, and the combination of pleural fluid ADA and T-SPOT. TB is of diagnostic value.

[Early secretory antigenic target protein-6/culture filtrate protein-10 fusion protein-specific Th1 and Th2 response and its diagnostic value in tuberculous pleural effusion].

OBJECTIVE: To detect the Th1 and Th2 cell percentage in pleural effusion mononuclear cells (PEMCs) stimulated by early secretory antigenic target protein-6 (ESAT-6)/culture filtrate protein-10 (CFP-10) fusion protein (E/C) with flow cytometry (FCM), and therefore to explore the local antigen specific Th1 and Th2 response and its diagnostic value in tuberculous pleuritis. METHODS: Forty patients with tuberculous pleural effusion and 30 patients with malignant pleural effusion were included in this study from Sep.2008 to Mar.2009. PEMCs were isolated and cryopreserved. After resuscitation, the cells were cultured with E/C (simultaneously with positive control and negative control), and antigen-specific Th1 and Th2 cells were detected with intracellular cytokine staining of FCM. Normal distribution data using t test, abnormal distribution data using Wilcoxon test. RESULTS: In the TB group,the medians (quartile range) of Th1 cells and Th1/Th2 ratio among PEMCs stimulated by ESAT-6/CFP-10 fusion protein were 3.06% (1.59%-6.92%) and 17 (7.38-35.53), significantly higher than those of the negative control [0.38% (0.02%-1.80%) and 3.59 (0.49-25.09)], the differences being statistically significant (Z = -5.345 and 3.314, P < 0.01). The percentage of Th2 cells [(0.22 ± 0.19)%] was also increased compared with that of the negative control [(0.10 ± 0.08)%], the difference being statistically significant (t = 4.108, P < 0.01). In the malignant effusion group, the medians (quartile range) of Th1 percentage and Th1/Th2 ratio were 0.12% (0.05%-0.39%) and 1.05 (0.25-2.52), which were significantly different as compared with those of the TB group (Z = -6.624 and -5.536, P < 0.01). The Th2 percentage in the 2 groups were (0.22 ± 0.19)% and (0.15 ± 0.02)%, respectively (t = 1.954, P > 0.05). The receiver operating characteristic curve indicated that the area under the curve (AUC), sensitivity, and specificity were 0.937, 85.4%, and 90.6% respectively for Th1 to diagnose tuberculous pleurisy. For Th1/Th2, the AUC, sensitivity, and specificity were 0.883, 81.5%, and 90.6% respectively. CONCLUSIONS: The feature of ESAT-6/CFP-10 fusion protein-specific Th1 and Th2 response in tuberculous pleurisy was a mixed reaction of Th1 and Th2 with Th1 predominance. Th1 percentage and Th1/Th2 ratio could be diagnostic indexes for identifying tuberculous from malignant pleural effusions.

An anti-LpqH human monoclonal antibody from an asymptomatic individual mediates protection against Mycobacterium tuberculosis.

Tuberculosis (TB) is an airborne disease caused by Mycobacterium tuberculosis (Mtb). Whilst a functional role for humoral immunity in Mtb protection remains poorly defined, previous studies have suggested that antibodies can contribute towards host defense. Thus, identifying the critical components in the antibody repertoires from immune, chronically exposed, healthy individuals represents an approach for identifying new determinants for natural protection. In this study, we performed a thorough analysis of the IgG/IgA memory B cell repertoire from occupationally exposed, immune volunteers. We detail the identification and selection of a human monoclonal antibody that exhibits protective activity in vivo and show that it targets a virulence factor LpqH. Intriguingly, protection in both human ex vivo and murine challenge experiments was isotype dependent, with most robust protection being mediated via IgG2 and IgA. These data have important implications for our understanding of natural mucosal immunity for Mtb and highlight a new target for future vaccine development.

[Comparison of two interferon-gamma release assays in the diagnosis of tuberculosis].

OBJECTIVE: To compare the diagnostic performance of A.TB, an ELISA-based interferon-gamma release assay (IGRA), and T-SPOT.TB, an ELISPOT-based IGRA, and therefore to evaluate the value of A.TB assay in the routine clinical practice. METHODS: From March to May of the year of 2011, 112 hospitalized patients were enrolled from 2 chest hospitals in Beijing and Harbin, including 75 cases in the TB group (43 male and 32 female) with the average age of (44 ± 18) years, spanning from 28 to 57 years, and 37 cases in the non-TB group (21 male and 16 female) with the average age of (54 ± 10) years, spanning from 24 to 82 years. During the same period, 34 healthy volunteers (4 male and 30 female), with the average age of (20 ± 0.6) years, spanning from 19 to 22 years, were recruited in Beijing Chest Hospital. A head-to-head comparison of the 2 IGRAs was performed on the 146 subjects to evaluate their overall diagnostic performance. Chi-square test or Fisher's exact test was used for statistical analysis of enumeration data. RESULTS: The sensitivity and specificity of A.TB were 81.3% (61/75, 95%CI = 72.5 - 90.2) and 83.1% (59/71, 95%CI = 74.4 - 91.8) respectively, compared to 90.7% (68/75, 95%CI = 84.1 - 97.3) and 78.9% (56/71, 95%CI = 69.4 - 88.4) for T-SPOT.TB. There was no significant difference in sensitivity or specificity (chi square values were 2.77 and 0.17 respectively, both P > 0.05). The area under the ROC curve was 0.90 (95%CI = 0.84 - 0.95) for A.TB and 0.91 (95%CI = 0.86 - 0.96) for T-SPOT.TB. The observation agreement between the 2 methods was 87.2% (123/141), with a kappa value of 0.74. T-SPOT.TB produced indeterminate results at a rate of 3.4% (5/146). CONCLUSIONS: There was comparable diagnostic performance between the 2 assays. However, when compared to T-SPOT.TB, the A.TB testing procedure, with less technical demand and without requirement of well-equipped lab, is simpler and the interpretation of results is less subjective.

A new method to rescue embryos contaminated by bacteria.

Objective: To report a case of successful pregnancy involving embryos that wereaffected by bacterial contamination. Design: A case report. Setting: Academic assisted reproductive center. Patients: A 31-year-old infertile patient with obstructed fallopian tubes facing bacterial contamination in her embryos during in vitro fertilization. Interventions: The zona pellucida (ZP) of the embryos that was contaminated by bacteria was removed by acidic Tyrode's solution. The ZP-free embryos were then cultured in a time-lapse culture dish with 1 zygote per well until day 5 when a single ZP-free blastocyst was selected for transfer. Main Outcome Measures: The rate of obtaining embryos without recurrence of bacterial contamination and the developmental potential of the embryos. Results: Twenty oocytes were retrieved and were coincubated with sperm in vitro overnight. A total of 9 zygotes with 2 pronuclei and 3 zygotes with 1 pronucleus were obtained. Unfortunately, all zygotes were contaminated by the Klebsiella pneumoniae bacteria. The ZP of 7 zygotes were removed using acidic Tyrode's solution (ZP-free group), whereas the remaining 5 zygotes and 3 metaphase II (MII) stage oocytes were washed with G-1 PLUS medium multiple times (washing treatment group). In the washing treatment group, all embryos experienced recontamination on day 2 and were dead by day 3. In the ZP-free group, 2 embryos were found to be recontaminated on day 2. The remaining 5 embryos that stayed uncontaminated were selected for blastocyst culture. On day 5, 2 of the cultured embryos developed into blastocysts. One blastocyst was transferred during the fresh cycle, and the other was vitrified. A single intrauterine gestation was confirmed 4 weeks after the transfer. At the time of writing this article, the patient was 30 weeks pregnant without any occurrence of intrauterine infection during pregnancy. Conclusions: Zona pellucida removal is a safe and effective method to rescue embryos contaminated with bacteria.

Rapid Detection of Mycobacterium tuberculosis in Pleural Fluid Using Resuscitation-Promoting Factor-Based Thin Layer Agar Culture Method.

BACKGROUND: Pleural tuberculous is difficult to diagnose. Culture is still considered the gold standard, especially in resource-limited settings where quick, cheap, and easy techniques are needed. The aim of the study was to evaluate resuscitation-promoting factors (Rpfs)-based thin layer agar (TLA) culture method for quick detection of Mycobacterium tuberculosis in pleural fluid. METHODS: Patients with suspected pleural TB were enrolled prospectively in our hospital, pleural fluid of all patients were collected, stained with Ziehl-Neelsen for acid-fast bacilli (AFB), cultured on Rpfs-TLA, TLA, and Löwenstein-Jensen (LJ) medium, and identified according to recommended procedures. RESULTS: A total of 137 suspected pleural TB were enrolled and categorized, including 103 pleural TB (49 confirmed and 54 probable pleural TB) and 34 non-TBP patients. The sensitivity of Rpfs-TLA for total pleural TB was 43.7% (34.5∼53.3%), higher than that of TLA 29.1% (21.2∼38.5%) and LJ 26.2% (18.7∼35.5%) (p < 0.01), and all specificity was 100% in the diagnosis of pleural TB. Median time to detection of a positive culture was 11.8 days (95% CI 10.4∼13.4) for Rpfs-TLA, 21.0 days (95% CI 19.1∼22.9) for TLA, and 30.5 days (95% CI 28.5∼32.5) for LJ (p < 0.001). CONCLUSION: Rpfs-TLA is an accurate, rapid, cheap, and easy culture method, which makes it promising for use in clinical laboratories.

Human antibodies targeting a Mycobacterium transporter protein mediate protection against tuberculosis.

Mycobacterium tuberculosis (Mtb) exposure drives antibody responses, but whether patients with active tuberculosis elicit protective antibodies, and against which antigens, is still unclear. Here we generate monoclonal antibodies from memory B cells of one patient to investigate the B cell responses during active infection. The antibodies, members of four distinct B cell clones, are directed against the Mtb phosphate transporter subunit PstS1. Antibodies p4-36 and p4-163 reduce Mycobacterium bovis-BCG and Mtb levels in an ex vivo human whole blood growth inhibition assay in an FcR-dependent manner; meanwhile, germline versions of p4-36 and p4-163 do not bind Mtb. Crystal structures of p4-36 and p4-170, complexed to PstS1, are determined at 2.1 Å and 2.4 Å resolution, respectively, to reveal two distinctive PstS1 epitopes. Lastly, a prophylactic p4-36 and p4-163 treatment in Mtb-infected Balb/c mice reduces bacterial lung burden by 50%. Our study shows that inhibitory anti-PstS1 B cell responses arise during active tuberculosis.

[Potential value of Mycobacterium tuberculosis RD1 region-encoded proteins in the diagnosis of tuberculosis].

Nine proteins encoded by Mycobacterium tuberculosis RD1 region are important protective antigens that become absent in long passaging of Mycobacterium tuberculosis. They only exist in pathogenic Mycobacteria and are absent in Bacille Calmette-Guerin and environmental Mycobacteria. With good immunogenicities, they may play an important role in the diagnosis and prevention of Mycobacterium tuberculosis. This article reviews recent studies on using RD1-encoded proteins as antigens in the diagnosis of active tuberculosis and tuberculous pleurisy.

A Two-Way Proteome Microarray Strategy to Identify Novel Mycobacterium tuberculosis-Human Interactors.

Tuberculosis (TB) is still a serious threat to human health which is caused by mycobacterium tuberculosis (Mtb). The main reason for failure to eliminate TB is lack of clearly understanding the molecular mechanism of Mtb pathogenesis. Determining human Mtb-interacting proteins enables us to characterize the mechanism and identify potential molecular targets for TB diagnosis and treatment. However, experimentally systematic Mtb interactors are not readily available. In this study, we performed an unbiased, comprehensive two-way proteome microarray based approach to systematically screen global human Mtb interactors and determine the binding partners of Mtb effectors. Our results, for the first time, screened 84 potential human Mtb interactors. Bioinformatic analysis further highlighted these protein candidates might engage in a wide range of cellular functions such as activation of DNA endogenous promoters, transcription of DNA/RNA and necrosis, as well as immune-related signaling pathways. Then, using Mtb proteome microarray followed His tagged pull-down assay and Co-IP, we identified one interacting partner (Rv0577) for the protein candidate NRF1 and three binding partners (Rv0577, Rv2117, Rv2423) for SMAD2, respectively. This study gives new insights into the profile of global Mtb interactors potentially involved in Mtb pathogenesis and demonstrates a powerful strategy in the discovery of Mtb effectors.

Brain-derived neurotrophic factor (BDNF) is expressed in buffalo (Bubalus bubalis) ovarian follicles and promotes oocyte maturation and early embryonic development.

Brain-derived neurotrophic factor (BDNF) has been discovered and characterized for several decades, yet its expression pattern in non-neuronal tissues like ovary and potential mechanism during oocyte maturation are still poorly understood. Thus the present study was devised to determine the expression pattern and mechanism of BDNF during buffalo oocyte maturation. The results revealed that BDNF was presented at different stages of buffalo ovarian follicles as well as during oocyte maturation and early embryo development. BDNF's receptor p75 was detected in granulosa cells, cumulus cells, oocytes, and early embryos, while another receptor neurotrophic tyrosine kinase receptor, type2 (NTRK2) was only identified in granulosa cells and cumulus cells. To determine the effect of BDNF on oocyte maturation and early embryo development, different concentrations (0, 1, 10, 100 ng/mL) of BDNF were added into the in vitro maturation media, respectively. It was divulged that 10 ng/mL BDNF promoted the in vitro maturation rate of buffalo oocytes and the blastocysts rate of embryos cultured in vitro (P < 0.05). Then through using NTRK2 inhibitor K-252a, we found BDNF and its receptor NTRK2 in cumulus cells played an essential role during oocyte maturation. Moreover, to further investigate the underlying mechanism by which BDNF enhances oocyte maturation, RT-qPCR was performed. 10 ng/mL BDNF treatment could decrease the expression level of apoptosis-related genes CCASP9, FAS, up-regulate the expression level of receptor gene NTRK2, cell proliferation-related genes CCNB1, PCNA, gap junction-related genes GJA4, GJA1 as well as cumulus cells expansion-related genes HAS2, PTX3 and TNFAIP6 (P < 0.05). Altogether, our results showed for the first time that BDNF was expressed throughout buffalo ovarian follicle development, oocyte maturation and early embryogenesis. Furthermore, BDNF treatment could improve the efficiency of buffalo oocyte maturation through regulating genes expression in cumulus cells and then promote early embryo development.

Application of the CRISPRi system to repress sepF expression in Mycobacterium smegmatis.

Despite technical advances in introducing genomic deletions and modulating gene expression, direct inactivation of essential genes in mycobacteria remains difficult. In this study, we described clustered regularly interspaced short palindromic repeat interference (CRISPRi) technology to repress the expression of sepF (MSMEG_4219) based on nuclease-deficient CRISPR-associated protein 9 (Cas9) and small guide RNA (sgRNA) specific to the target sequence in Mycobacterium smegmatis. Using this CRISPRi approach, we achieved the repression of sepF by up to 98% in M. smegmatis without off-target effects. The depleted Msm_sepF strains resulted in growth and morphology changes including elongated, filamentous and branched bacterial cells, but the levels of the interacting partners ftsZ and murG were not modified in M. smegmatis. The sepF gene was proven to be an essential gene in M. smegmatis. This study provided an improved and detailed technical procedure for the application of CRISPRi technology in mycobacteria, and this approach was demonstrated to be a simple and efficient tool for regulating the expression of essential genes in M. smegmatis.

.Expression and purification of Mycobacterium tuberculosis MPT83 protein and its application in tuberculosis immunological diagnosis / 中国热带医学

@#Abstract: Objective To express and purify MPT83 protein of Mycobacterium tuberculosis and evaluate its application value in immunological diagnosis of tuberculosis (TB) using clinical samples. Methods Using Mycobacterium tuberculosis (Mtb) H37Rv genome as the template, Mtb mpt83 gene was amplified by PCR and connected to PET-21a (+) to construct prokaryotic expression vector, and then transferred into E.coli DH5α. The positive colonies were picked out and retained. The recombinant plasmid pET-mpt83 of the strain with positive colony PCR was extracted, identified by double digestion, and the samples of the positive colonies were sent for sequencing. The correctly sequenced plasmids then were transferred into BL21 competent cells for induction, expression and purification with nickel column affinity chromatography. The purified products were identified by 12 alkyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Mouse polyclonal antiserum was prepared by immunizing mice with purified protein. 8 patients clinically diagnosed as tuberculosis pleural effusion (TB group) and 8 adenocarcinomas patients (CA group) were enrolled and their pleural effusion and plasma were collected. 8 healthy people (HC group) were enrolled as the control group and their plasma were collected. An indirect ELISA was used to detect the level of specific antibodies recognizing MPT83 protein in the samples. Results Mtb MPT83 protein was successfully expressed and purified. The serum titer of MPT83 mouse polyclonal antibody was as high as 1∶1 280 000. The plasma levels of MPT83 antigen specific antibodies in TB group were significantly higher than those in HC group (P<0.05), while the plasma levels of MPT83 antigen specific antibodies in CA group were not significantly different from those in HC group (P>0.05). Compared with the HC group, there was no significant difference in pleural fluid in both the TB and CA groups (P>0.05). The ROC curve was used to analyze the OD values of plasma in TB group and HC group, and the area under the curve was greater than 0.7, showing high diagnostic efficacy. Conclusion　MPT83 protein has high antigen specificity and immunogenicity, which has great application value in the immunological diagnosis of tuberculosis.

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.

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.

Association of Mannose-binding Lectin Polymorphisms with Tuberculosis Susceptibility among Chinese.

Tuberculosis (TB) is caused by infection of Mycobacterium tuberculosis. Host genetic variability is an important determinant of the risk of developing TB in humans. Although the association between MBL2 polymorphisms and TB has been studied in various populations, the results are controversial. In this study four functional single-nucleotide polymorphisms (SNPs, H/L, X/Y, P/Q and A/B) across the MBL2 gene were genotyped by direct DNA sequencing of PCR products in a case-control population of Chinese Han origin, consisting of 1,020 patients with pulmonary TB and 1,020 controls. We found that individuals carrying variant allele at A/B (namely BB or AB genotypes) was associated with increased susceptibility to TB (odds ratios [OR] = 1.57, 95% confidence interval [CI] 1.30-1.91, P = 1.3 × 10-6). Additionally, LYPB haplotype showed a significant association with increased risk of TB (OR = 1.54, 95% CI 1.27-1.87, P = 4.2 × 10-6; global haplotype association P = 3.5 × 10-5). Furthermore, individuals bearing low- or medium- MBL expression haplotype pairs had an increased risk of TB (OR = 1.56, 95% CI 1.29-1.90, P = 1.4 × 10-6). Thus, the reduced expression of functional MBL secondary to having MBL2 variants may partially mediate the increased susceptibility to TB risk.

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.

Interferon-gamma release assay performance of pleural fluid and peripheral blood in pleural tuberculosis.

BACKGROUND: The diagnosis of pleural tuberculosis (TB) remains to be difficult. Interferon-gamma release assay (IGRA) is a promising method for diagnosing TB in low TB burden countries. The release of interferon-gamma (IFN-γ) by T lymphocytes increases at a localized site of infection with Mycobacterium tuberculosis antigen. This study aimed to examine the clinical accuracy of T-SPOT.TB on pleural fluid and peripheral blood for the diagnosis of pleural TB in high TB burden country. METHODS: 168 subjects with pleural effusion were enrolled prospectively and examined with T-SPOT.TB on pleural fluid and peripheral blood samples simultaneously. RESULTS: The receiver operating characteristic (ROC) curve and cut-off value of pleural fluid T-SPOT.TB was established according to spot forming cells (SFC) between culture/biopsy-confirmed pleural TB group and no pleural TB group. The sensitivity of pleural fluid T-SPOT.TB and peripheral blood T-SPOT.TB was similar (96.3% and 92.7%, respectively) (P= 0.691). In contrast, the specificity of pleural fluid T-SPOT.TB (94.5%) was significantly higher than that of peripheral blood T-SPOT.TB (76.1%) (P=0.002). 2% (2/98) of pleural fluid T-SPOT.TB results were indeterminate. CONCLUSION: The diagnostic accuracy of peripheral blood T-SPOT.TB is low in high TB burden countries due to latent tuberculosis infection. Pleural fluid T-SPOT.TB is a relatively useful and supplementary test to explore pleural TB in high TB burden countries, but its diagnostic accuracy needs to be validated in further large scale research.

Application of hyperbranched rolling circle amplification for direct detection of mycobacterium tuberculosis in clinical sputum specimens.

BACKGROUND: Global tuberculosis (TB) control is encumbered by the lack of a rapid and simple detection method for diagnosis, especially in low-resource areas. An isothermal amplification method, hyperbranched rolling circle amplification (HRCA), was optimized to detect Mycobacterium tuberculosis (Mtb) in clinical sputum specimens. METHODS: A clinical validation study was performed to assess the diagnostic accuracy of HRCA. In order to analyze the detection limit of HRCA under optimal conditions, the method was initially used to detect purified H37Rv strain DNA and culture suspensions. Next, three strains of Mycobacterium tuberculosis complex (MTC) and eight strains of non-tuberculosis mycobacterium (NTM) were analyzed in order to evaluate specificity. Sputum specimens from 136 patients with diagnosed pulmonary TB, 38 lung cancer patients, and 34 healthy donors were tested by HRCA to validate the clinical application of HRCA for the rapid detection of Mtb. RESULTS: The detection limit of HRCA for purified H37Rv DNA and culture suspensions was 740 aM and 200cfu/ml, respectively. The results of all MTC strains were positive in contrast to the NTM specimens which were all negative. The detection sensitivity for the 136 sputum specimens from TB patients was 77.2% (105/136), which was slightly lower than that of quantitative real-time PCR(79.4%, 108/136) and culture (80.9%,110/136). The sensitivity of all three methods was statistically higher than smear microscopy (44.9%, 61/136). The overall specificity of HRCA was 98.6% (71/72) which was similar to that of quantitative real-time PCR (qRT-PCR) and smear/culture methods (100%, 72/72). CONCLUSIONS: Use of the HRCA assay for detection of Mtb within clinical sputum specimens was demonstrated to be highly sensitive and specific. Moreover, the performance of HRCA is simple and cost-effective compared with qRT-PCR and is less time consuming than culture. Therefore, HRCA is a promising TB diagnostic tool that can be used routinely in low-resource clinical settings.

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.