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Objectives: Distinguishing between active tuberculosis (ATB) and latent tuberculosis infection (LTBI) remains challenging.Methods: The modified T-SPOT.TB assay was performed in 499 participants (243 ATB and 256 LTBI) and another 322 participants (162 ATB and 160 LTBI) who were diagnosed in Qiaokou (training) and Caidian (validation) cohort respectively.Results: The mean spot sizes (MSS) of early secreted antigenic target 6 (ESAT-6) spot-forming cells (SFC) of T-SPOT.TB assay in ATB patients was significantly higher than that in LTBI individuals. 1.0 × 105 was the optimal number of cells added to phytohaemagglutinin (PHA) well for obtaining more accurate TB-specific antigen to phytohaemagglutinin (TBAg/PHA) ratio. The area under the curve of the diagnostic model by combination of ESAT-6 SFC MSS and modified TBAg/PHA ratio in distinguishing ATB from LTBI was 0.959 in training cohort, with a sensitivity of 90.12% and a specificity of 91.02% when a cutoff value of 0.46 was used. This diagnostic model showed similar performance in the validation cohort. The area under the curve, sensitivity, and specificity were 0.962, 93.21%, and 90.00%, respectively. Further flow cytometry analysis showed that ESAT-6 stimulation induced a significantly higher mean fluorescence intensity of IFN-γ+ cells in lymphocytes compared with culture filtrate protein 10 (CFP-10) stimulation. In contrast, CFP-10 stimulation induced a significantly higher percentage of IFN-γ+ cells in lymphocytes compared with ESAT-6 stimulation.Conclusions: The combination of the MSS of ESAT-6 SFC and the modified TBAg/PHA ratio of T-SPOT.TB assay showed great value in discriminating ATB from LTBI. [ABSTRACT FROM AUTHOR]

Nanopore sensors have shown great utility in nucleic acid detection and sequencing approaches. Recent studies also indicate that current signatures produced by peptide-nanopore interactions can distinguish high purity peptide mixtures, but the utility of nanopore sensors in clinical applications still needs to be explored due to the inherent complexity of clinical specimens. To fill this gap between research and clinical nanopore applications, we describe a methodology to select peptide biomarkers suitable for use in an immunoprecipitation-coupled nanopore (IP-NP) assay, based on their pathogen specificity, antigenicity, charge, water solubility and ability to produce a characteristic nanopore interaction signature. Using tuberculosis as a proof-of-principle example in a disease that can be challenging to diagnose, we demonstrate that a peptide identified by this approach produced high-affinity antibodies and yielded a characteristic peptide signature that was detectable over a broad linear range, to detect and quantify a pathogen-derived peptide from digested human serum samples with high sensitivity and specificity. This nanopore signal distinguished serum from a TB case, non-disease controls, and from a TB-case after extended anti-TB treatment. We believe this assay approach should be readily adaptable to other infectious and chronic diseases that can be diagnosed by peptide biomarkers. To fill the gap between the nanopore-based peptide sensors on bench and their application in clinic. We describe a standard workflow identifies peptide targets suitable for use in nanopore-based peptide assays in clinical samples, to detect and quantify a pathogen-derived peptide from digested human serum samples with high sensitivity and specificity. [Display omitted] • A standard workflow identifies peptide targets suitable for use in nanopore assays. • Immunoprecipitation-coupled nanopore peptide assays can analyze clinical samples. • Nanopore-read immunoassays have good target sensitivity and specificity. • Nanopore peptide results can distinguish treated and untreated cases and controls. [ABSTRACT FROM AUTHOR]

Diagnosing tuberculous pleurisy (TP) remains a clinical challenge and the best method to diagnose it is controversial. Although several studies have investigated the performance of pleural fluid (PF) T-SPOT for pleural tuberculosis (plTB) diagnosis, the heterogeneity of its accuracy exists. Therefore, we performed an updated meta-analysis of the existing evidence on the utility of PF T-SPOT to diagnose TP. PubMed and EmBase were searched for relevant English articles up to July 29, 2019. Statistical analysis was performed using Stata, Revman, and Meta-Disc. Pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR) were determined. Summary receiver operating characteristic (SROC) curves and the area under the curve (AUC) were used to summarize the overall diagnostic performance. A total of 13 studies (997 patients with TP and 656 patients without TP) were identified and enrolled to meta-analysis, giving the following pooled values for diagnostic accuracy of PF T-SPOT: sensitivity, 0.91 (95% CI, 0.89–0.92, I 2 = 80.9%); specificity, 0.88 (95% CI, 0.86–0.91, I 2 = 87.3%); PLR, 6.28 (95% CI, 2.88–13.69, I 2 = 93.3%); NLR, 0.12 (95% CI, 0.07–0.21, I 2 = 84.9%); DOR, 59.74 (95% CI, 24.13–147.93, I 2 = 78.3%); and the area under the SROC curve, 0.95 (95% CI, 0.93–0.97). Our meta-analysis suggests that PF T-SPOT has important diagnostic value for plTB. However, the standardization of the operating procedure needs to be further promoted, which would make the results more credible. [ABSTRACT FROM AUTHOR]

• PF T-SPOT shows good diagnostic utility for TP diagnosis. • The performance of PF T-SPOT is comparable to that of IFN-γ in diagnosing TP. • The combination of PF T-SPOT and IFN-γ can show a better performance in diagnosing TP. Early and accurate diagnosis of tuberculous pleurisy (TP) remains a challenge. The aim of the present study is to evaluate the performance of the pleural fluid (PF) T-SPOT and interferon-gamma (IFN-γ) for TP diagnosis in high tuberculosis (TB) burden settings. In total, 214 and 217 subjects suspected of TP were prospectively enrolled in the Wuhan (training) cohort and Changchun (validation) cohort, respectively. All patients were examined with PF T-SPOT, IFN-γ, and other traditional tests simultaneously. The receiver-operating characteristic (ROC) curve analysis showed that the area under the curve (AUC), sensitivity, and specificity of TB-specific antigen (TBAg) spot-forming cells (SFC) (the larger of early secreted antigenic target 6 and culture filtrate protein 10 SFC in PF T-SPOT assay) for TP diagnosis were 0.972, 92.86%, and 92.16%, respectively, with a cutoff value of 35 in the Wuhan cohort. Meanwhile, when a threshold value of 95 ng/mL was set, the AUC, sensitivity, and specificity of IFN-γ to diagnose TP were 0.951, 86.61%, and 90.20%, respectively. Moreover, the diagnostic model based on the combination of TBAg SFC and IFN-γ showed an AUC of 0.983 for differentiating TP from non-TP, with 95.54% sensitivity and 95.10% specificity when a cutoff value of 0.32 was used in the Wuhan cohort. Excellent diagnostic accuracy was also observed in the Changchun cohort. When applying the cutoff value obtained from the Wuhan cohort, the AUC, sensitivity, and specificity of the diagnostic model were 0.995, 95.08%, and 97.89%, respectively. The performance of PF T-SPOT was comparable to IFN-γ in diagnosing TP. However, using the diagnostic model established by the combination of these two assays can achieve a more accurate diagnosis of TP. [ABSTRACT FROM AUTHOR]

• Iron metabolism indexes could be used to distinguish between active tuberculosis (ATB) and latent tuberculosis infection (LTBI). • The TB-specific antigen/phytohemagglutinin (TBAg/PHA) ratio could serve as an indicator for discriminating ATB from LTBI. • A combination of iron metabolism indexes and the TBAg/PHA ratio could yield a better value in differentiating ATB from LTBI. Discriminating active tuberculosis (ATB) from latent tuberculosis infection (LTBI) remains challenging. This study aimed to investigate a diagnostic model based on a combination of iron metabolism and the TB-specific antigen/phytohemagglutinin ratio (TBAg/PHA ratio) in T-SPOT.TB assay for differentiation between ATB and LTBI. A total of 345 participants with ATB (n = 191) and LTBI (n = 154) were recruited based on positive T-SPOT.TB results at Tongji hospital between January 2017 and January 2020. Iron metabolism analysis was performed simultaneously. A diagnostic model for distinguishing ATB from LTBI was established according to multivariate logistic regression. The TBAg/PHA ratio showed 64.00% sensitivity and 90.10% specificity in distinguishing ATB from LTBI when a threshold of 0.22 was used. All iron metabolism biomarkers in the ATB group were significantly different from those in the LTBI group. Specifically, serum ferritin and soluble transferrin receptor in ATB were significantly higher than LTBI. On the contrary, serum iron, transferrin, total iron binding capacity, and unsaturated iron binding capacity in ATB were significantly lower than LTBI. The combination of iron metabolism indicators accurately predicted 60.00% of ATB cases and 91.09% of LTBI subjects, respectively. Moreover, the combination of iron metabolism indexes and TBAg/PHA ratio resulted in a sensitivity of 88.80% and specificity of 90.10%. Furthermore, the performance of models established in the Qiaokou cohort was confirmed in the Caidian cohort. The data suggest that the combination of iron metabolism indexes and TBAg/PHA ratio could serve as a biomarker to distinguish ATB from LTBI in T-SPOT-positive individuals. [ABSTRACT FROM AUTHOR]

• Novel coronavirus pneumonia patients are frequently encountered in the sixth and seventh decades of life • Significantly decreased white blood cells, alkaline phosphatase and d-dimer were observed in novel coronavirus pneumonia patients compared with influenza patients • Lactate dehydrogenase, erythrocyte sedimentation rate and fibrinogen were significantly increased in novel coronavirus pneumonia patients compared with those in influenza patients • An optimal diagnostic model with moderate value was established based on the combination of 18 biomarkers from routine laboratory tests to discriminate novel coronavirus pneumonia patients from influenza patients The differential diagnosis between novel coronavirus pneumonia patients (NCPP) and influenza patients (IP) remains a challenge in clinical practice. Between January 2018 and March 2020, 1,027 NCPP and 1,140 IP were recruited from Tongji hospital. Routine blood examination, biochemical indicators and coagulation function analysis were simultaneously performed in all participants. There was no sex predominance in NCPP. The NCPP were frequently encountered in the sixth and seventh decades of life. The mean age of NCPP (56 ± 16 years) was higher than IP (47 ± 17 years), but without statistical difference. Although most results of routine laboratory tests between NCPP and IP had no significant differences, some laboratory tests showed an obvious change in NCPP. It was observed that NCPP had significantly decreased white blood cells, alkaline phosphatase and d-dimer compared with IP. However, the results of lactate dehydrogenase, erythrocyte sedimentation rate and fibrinogen were significantly increased in NCPP compared with IP. The diagnostic model based on a combination of 18 routine laboratory indicators showed an area under the curve of 0.796 (95% CI, 0.777–0.814), with a sensitivity of 46.93% and specificity of 90.09% when using a cut-off value of 0.598. Some routine laboratory results had statistical difference between NCPP and IP. A diagnostic model based on a combination of routine laboratory results provided an adjunct approach in the differential diagnosis between NCPP and IP. [ABSTRACT FROM AUTHOR]

Summary Objectives The results of the T-SPOT.TB (T-SPOT) assay are reduced in immunocompromised patients with active tuberculosis (ATB), and it is difficult using T-SPOT results to distinguish ATB from latent tuberculosis infection (LTBI) in this condition. The aim of this study was to determine the performance of the TBAg/PHA ratio in T-SPOT assay in the diagnosis of ATB in immunocompromised patients. Methods One hundred and forty three immunocompromised ATB patients and 124 LTBI individuals were diagnosed according to conventional tests and T-SPOT assay. Results The results of T-SPOT assay are of no value in the diagnosis of ATB in immunocompromised patients. However, the number of phytohaemagglutinin (PHA) spot-forming cells (sfc) in T-SPOT assay was substantially decreased in immunocompromised ATB patients compared with that in LTBI individuals. Receiver operating characteristic (ROC) analysis revealed that a further calculation of the TBAg/PHA ratio (the larger of the ESAT-6/PHA and CFP-10/PHA) showed a better performance in distinguishing these two diseases. Using the threshold value of 0.316, the sensitivity and specificity for distinguishing immunocompromised ATB patients from LTBI individuals were respectively 79.21 and 94.05%. Conclusions Our findings suggest that the TBAg/PHA ratio might have some significance for the diagnosis of TB disease in immunocompromised patients. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells play a key role in host defense against microbial pathogens. Establishing the reference intervals (RIs) of NK cell functions would be valuable in assessing the immune status of hosts. We evaluated the NK cell activity in healthy adults. We further established and validated the RIs of representative NK cell functions. Flow cytometry was used to evaluate the cytokine production and CD107a degranulation of NK cells. Levels of soluble IFN-γ in the culture supernatants were evaluated by ELISA. Our results demonstrated that the intracellular IFN-γ production of NK cells was positively correlated with CD107a expression and soluble IFN-γ levels. There were no significant differences in NK cell functions between different age and gender groups. The mean values and RIs of representative NK cell functions are as following: IFN-γ + NK cells (%): 28.09 (11.3–51.95); CD107a + NK cells (%): 17.90 (9.852–27.56); soluble IFN-γ (pg/ml): 330.4 (41.38–717.8). In addition, the intracellular IFN-γ production and degranulation activity of NK cells in patients with colorectal cancer were significantly lower than that in healthy adults. Our study has established the RIs of NK cell functions in healthy adults, which might be used for monitoring the immune status of the hosts. [ABSTRACT FROM AUTHOR]