Mycobacterium tuberculosis latency-associated antigen Rv1733c SLP improves the accuracy of differential diagnosis of active tuberculosis and latent tuberculosis infection.

BACKGROUND: Differential diagnosis of active tuberculosis (ATB) and latent tuberculosis infection (LTBI) has been a challenge for clinicians in high TB burden countries. The purpose of this study was to improve the accuracy of differential diagnosis of ATB and LTBI by using fluorescent immunospot (FluoroSpot) assay to detect specific Th1 cell immune responses. The novel mycobacterium tuberculosis (MTB) latency-associated antigens Rv1733c and synthetic long peptides derived from Rv1733c (Rv1733c SLP) were used based on virulence factors early secreting antigen target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10). METHODS: Fifty-seven ATB cases, including 20 pathogen-confirmed ATB and 37 clinically diagnosed ATB, and 36 LTBI cases, were enrolled between January and December 2017. FluoroSpot assay was used to detect the interferon γ (IFN-γ) and interleukin 2 (IL-2) secreted by the specific T cells after being stimulated with MTB virulence factors ESAT-6 and CFP-10, MTB latency-associated antigens Rv1733c and Rv1733c SLP. The receiver operating characteristic (ROC) curve was used to define the best cutoff value of latency-associated antigens in the use of differentiating ATB and LTBI. The sensitivity, specificity, predictive value, and likelihood ratio of ESAT-6 and CFP-10-FluoroSpot combined with latency-associated antigen in the differential diagnosis of ATB and LTBI were also calculated. RESULTS: Following the stimulation with Rv1733c and Rv1733c SLP, the frequency of single IL-2-secreting T cells stimulated by Rv1733c SLP had the largest area under the ROC curve, which was 0.766. With a cutoff value of 1 (spot-forming cells [SFCs]/2.5 × 105 peripheral blood mononuclear cells) for frequency, the sensitivity and specificity of distinguishing ATB from LTBI were 72.2% and 73.7%, respectively. ESAT-6 and CFP-10-FluoroSpot detected the frequency and proportion of single IFN-γ-secreting T cells; the sensitivity and specificity of distinguishing ATB from LTBI were 82.5% and 66.7%, respectively. Combined with the frequency of single IL-2-secreting T cells stimulated by Rv1733c SLP on the basis of ESAT-6 and CFP-10-FluoroSpot, the sensitivity and specificity increased to 84.2% and 83.3%, respectively. CONCLUSION: Rv1733c SLP, combined with ESAT-6 and CFP-10, might be used as a candidate antigen for T cell-based tuberculosis diagnostic tests to differentiate ATB from LTBI.

Utility of interferon gamma/tumor necrosis factor alpha FluoroSpot assay in differentiation between active tuberculosis and latent tuberculosis infection: a pilot study.

BACKGROUND: The differential diagnosis of active tuberculosis (ATB) and latent tuberculosis infection (LTBI) remains challenging in clinical practice. We aimed to evaluate the diagnostic accuracy of the IFN-γ/TNF-α FluoroSpot assay for differentiating ATB from LTBI. METHODS: We conducted a pilot study of case-control design, using the FluoroSpot assay to simultaneously detect IFN-γ and TNF-α secretion at the single-cell level. The frequencies of antigen-specific single TNF-α-, total TNF-α-, single IFN-γ-, total IFN-γ- and dual IFN-γ/TNF-α-secreting T cells were detected. The optimal cutoffs value of frequencies for differentiating ATB from LTBI were determined according to receiver operating characteristic curve analysis. The sensitivity, specificity, predictive values (PV) and likelihood ratios (LR) of the FluoroSpot assay were calculated. RESULTS: Thirty patients diagnosed microbiologically with ATB, 36 healthcare workers with LTBI and 36 healthy controls were enrolled. After stimulated by ESAT-6 or CFP-10 peptides, the median frequencies of single TNF-α-, total TNF-α-, single IFN-γ-, total IFN-γ- and dual IFN-γ/TNF-α-secreting T cells in ATB patients were all significantly higher than those in LTBI and HC groups (P < 0.01). The frequencies of total IFN-γ-secreting T cells detected by FluoroSpot assay correlated significantly with those of T-SPOT.TB (r = 0.910 for ESAT-6, P < 0.001, r = 0.845 for CFP-10, P < 0.001). After stimulated by ESAT-6 peptides, with total TNF-α-secreting T cells frequencies at a cut off value of 21 iSFCs/250,000 PBMCs, the sensitivity, specificity, PLR, NLR, PPV, NPV of IFN-γ/TNF-α FluoroSpot assay in differentiating ATB from LTBI were 96.7% (95%CI, 82.8-99.9%), 94.3% (95%CI, 80.8-99.3%), 16.92 (95%CI, 4.40-65.08), 0.04 (95%CI, 0.01-0.24), 93.6% (95%CI,78.6-99.2%) and 97.1% (95%CI, 84.7-99.9%), respectively. With the frequencies of total TNF-α- and total IFN-γ-secreting T cells stimulated by ESAT-6 peptides combined, the specificity was increased to 97.1%, and the positive likelihood ratio to 31.5. The combination with CFP-10 might not improve the diagnostic accuracy of the ESAT-6 for differentiating ATB from LTBI. CONCLUSIONS: IFN-γ/TNF-α FluoroSpot assay might have potential to help differentiate ATB from LTBI, but the findings need to be further verified by cross-sectional or prospective cohort studies.

Application of IFN-γ/IL-2 FluoroSpot assay for distinguishing active tuberculosis from non-active tuberculosis: A cohort study.

Currently available Interferon-γ release assay cannot reliably differentiate active TB (ATB) from non-active TB (non-ATB). This study aimed to evaluate the diagnostic accuracy of the IFN-γ/IL-2 FluoroSpot assay, which can simultaneously detect IFN-γ and IL-2 secretion, for differentiating ATB from non-ATB. 191 suspected ATB patients with positive T-SPOT.TB results were consecutively recruited. 64 (33.5%) participants had ATB, including 22 (34.4%) microbiologically or histologically confirmed TB and 42 (65.6%) clinically diagnosed TB. 119 (62.3%) cases were non-ATB and 8 (4.2%) were clinically indeterminate. After being stimulated with ESAT-6 and CFP-10 antigens, the median frequency and proportion of IFN-γ+IL-2- T cells were significantly higher in the ATB group than the non-ATB group (P < .001). The areas under the ROC curves of IFN-γ+IL-2- T cells were larger than those of total IFN-γ+ T cells (0.788 vs. 0.739, p = .323). With a cutoff value of 25 SFCs/250,000 PBMCs for frequency, sensitivity and specificity of this assay were 73.4% and 69.8% respectively. When combining the frequency and proportions of IFN-γ+IL-2- T cells, the sensitivity and specificity were increased to 95.3% in parallel testing and 83.2% in serial testing respectively. In conclusion, IFN-γ/IL-2 FluoroSpot assay is conducive for the diagnosis of ATB in patients with positive T-SPOT.TB results.

Prevalence and influencing factors of the high nil-control spot count in T-SPOT.TB: A matched case-control study.

BACKGROUND: T-SPOT.TB may yield indeterminate results, including high nil responses and insufficient mitogen responses. We explored the incidence and risk factors of high nil responses. METHODS: A 1:1 matched case-control study of patients who underwent T-SPOT.TB tests in Peking Union Medical College Hospital from Jan 1, 2015 to Apr 30, 2017 was conducted. High nil responses were defined as >10 spots in negative control wells. Cases and controls were matched based on when the tests were performed. Demographic, clinical and laboratory data were obtained from the Medical Record System. RESULTS: A total of 644 out of 36,316 (1.76%, 95% CI: 1.63%-1.90%) patients presented with high nil responses (280 cases and 280 controls were enrolled). Multivariate analysis revealed that male (OR = 1.882, 95% CI: 1.222-2.899), Behcet's disease (OR = 7.764, 95% CI: 1.714-35.167), heavy use of corticosteroids within a month (OR = 0.357, 95% CI: 0.138-0.921, for >1000 mg group) and hypoalbuminemia (OR = 0.385, 95% CI: 0.241-0.615) are significantly associated with high nil responses. CONCLUSIONS: High nil responses in T-SPOT.TB assays are quite rare. Male gender and Behcet's disease are suggested as independent risk factors, while recent excessive use of corticosteroids and hypoalbuminemia seem to be independent protective factors.

Studies on pharmacokinetics and bioavailability of aminophylline in partridge chickens.

Veterinary medicine plays a significant role in the development of animal husbandry. Drugs residual in food would follow the food-chain coming into human body, which might bring hidden dangers to people. Chicken is the prime source of meat food, whose quality is important for our life and health. Therefore, it is necessary to realize the withdrawal period and establish an efficient, sensitive and accurate method for monitoring the metabolic process of drugs in chicken body. In this paper, the pharmacokinetics of aminophylline in partridge chicken after intravenous and oral administration was investigated using a sensitive high-performance liquid chromatography method. Plasma concentration-time profiles of aminophylline were analyzed by a non-compartmental model using Topfit 2.0. Following intravenous and oral administration, the peak concentrations (C max) were found to be (16.5 ± 3.0) µg/mL at (0.08 ± 0) h and (7.4 ± 1.5) µg/mL at (1.83 ± 1.11) h, respectively. The elimination half-time (t 1/2) after intravenous and oral administration were, respectively, (13.1 ± 4.17) h and (11.65 ± 1.14) h. Areas under the plasma concentration-time curve (AUC) were (209.6 ± 22.8) µg h mL(-1)(AUC0-t ) and (219.5 ± 28.3) µg h mL(-1) (AUC0→∞ ) after intravenous, and (165.1 ± 37.0) µg h mL(-1)(AUC0-t ) and (179.3 ± 35.6) µg h mL(-1) (AUC0→∞ ) after oral administration. Mean retention time (MRT) after intravenous and oral administration were, respectively, (14.06 ± 0.86) and (15.27 ± 0.62) h. The total clearance rates (CLtol) were (0.77 ± 0.10) mL min(-1) kg(-1) of intravenous and (0.97 ± 0.20) mL min(-1) kg(-1) of oral administration. The apparent distribution volume (V d) was (0.87 ± 0.27) and (0.97 ± 0.20) L kg(-1), respectively, for intravenous and oral administration. The absolute bioavailability (F) after oral administration was (83.1 ± 11.7) %. The results showed that aminophylline in partridge chickens had a longer elimination half-time, a smaller clearance rate, as well as a higher absolute bioavailability for oral administration. Therefore, aminophylline in partridge chickens produced a long healing efficacy and oral administration can achieve a good absorption which could meet the requirement.