Utility of rapid on-site evaluation during bronchoscopy in the diagnosis of pulmonary tuberculosis.

Introduction：The diagnostic value of rapid on-site evaluation (ROSE) in bronchoscopy for lung tumors has been widely researched. However, the diagnostic efficacy of ROSE for pulmonary tuberculosis has not been extensively assessed yet. This study aimed to examine the value of ROSE in diagnosing pulmonary tuberculosis during bronchoscopy, and the relationship between ROSE cytology patterns and acid-fast bacilli (AFB) smears and mycobacterial cultures. Methods：A retrospective study was conducted at a single respiratory endoscopy centre, including 418 patients under clinical or radiological suspicion of having pulmonary tuberculosis who underwent bronchoscopy. In addition to the use of ROSE and definitive cytology, material obtained by aspiration/lavage or brushing was sent for AFB smear and mycobacterial culture. If histopathological examination was required, endobronchial biopsy (EBB), transbronchial lung biopsy (TBLB) and transbronchial needle aspiration (TBNA) were performed at the discretion of the clinician. A composite reference standard (CRS) was used as the diagnostic gold standard for this study. The diagnosis obtained by ROSE was compared with the final diagnosis. Results：Of the 418 patients studied, 282 (67.5%) were diagnosed on the basis of bronchoscopic findings, as follows: pulmonary tuberculosis, in 238 (84.4%); Non-tuberculosis (Non-TB) ，in 44 (15.6%). In 238 pulmonary tuberculosis patients, ROSE cytology showed granulomas without necrosis were observed in 107 cases, granulomas and necrosis in 51 cases, caseous necrosis only in 25 cases, and non-specific inflammation in 55 cases. For the diagnosis of tuberculosis according to CRS, ROSE showed the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) was 76.9%, 68.2%, 92.9% and 35.3%, respectively. The positivity rate for bacterial detection by acid-fast staining and culture at bronchoscopy was 51.7%. The cytological pattern showed a higher detection rate for bacteria in cases of necrosis. Discussion：The application of rapid on-site evaluation (ROSE) during bronchoscopy is a straightforward procedure that delivers an immediate and precise assessment regarding the adequacy of collected samples, enabling a preliminary diagnosis of pulmonary tuberculosis. ROSE has exhibited a higher sensitivity in detecting pulmonary tuberculosis compared to microbiological examinations. In addition, the cytological presentation of ROSE tends to show a higher positivity rate for microbiological testing in caseous necrosis. Therefore, samples with these characteristics should be prioritised for microbiological examination after on-site evaluation.

Toehold-Containing Three-Way Junction-Initiated Multiple Exponential Amplification and CRISPR/Cas14a Assistant Magnetic Separation Enhanced Visual Detection of Mycobacterium Tuberculosis.

Rapid and simple nucleic acid detection is significant for disease diagnosis and pathogen screening, especially under specific conditions. However, achieving highly sensitive and specific nucleic acid detection to meet the time and equipment demand remains technologically challenging. In this study, we proposed a magnetic separation enhanced colorimetry biosensor based on a toehold-containing three-way junction (TWJ) induced multiple isothermal exponential amplification and the CRISPR/Cas14a (C-TEC) biosensor. The TWJ template was designed as a Y-X-Y structure. In the presence of the target, the formation of toehold-containing TWJ complex induced primer extension, leading to the generation of amplified single-stranded DNA; this amplified DNA could then bind to either the free TWJ template for EXPAR reaction or the toehold of the TWJ complex for toehold-mediated strand displacement, thereby enabling the recycling of the target. The amplification products could trigger CRISPR/Cas14a for efficient trans-cleavage and release the magnetically bound gold nanoparticle probes for colorimetry detection. Using Mycobacterium tuberculosis 16S rDNA as the target, the proposed C-TEC could detect 16S rDNA down to 50 fM by the naked eye and 20.71 fM by UV-vis detector at 520 nm within 90 min under optimal conditions. We successfully applied this biosensor to clinical isolates of Mycobacterium tuberculosis. In addition, the C-TEC biosensor also showed feasibility for the detection of RNA viruses. In conclusion, the proposed C-TEC is a convenient, fast, and versatile platform for visual detection of pathogen DNA/RNA and has potential clinical applications.

Estradiol inhibits autophagy of Mycobacterium tuberculosis­infected 16HBE cells and controls the proliferation of intracellular Mycobacterium tuberculosis.

Tracheobronchial tuberculosis (TBTB) is most common in young, middle­aged females. Despite adequate anti­tuberculosis therapy, >90% of patients develop tracheobronchial stenosis, which has a high rate of resulting in disability. The present study aimed to explore the effect of estradiol on the development of TBTB. Estrogen receptor (ER) expression in granulomatous tissue was assessed via immunofluorescence. In order to determine whether estrogen affected the proliferation of intracellular Mycobacterium tuberculosis (Mtb), 16HBE cells were infected with Mtb in vitro, followed by estradiol treatment. Intracellular Mtb was quantified via colony counting. The effect of estradiol on autophagy of infected 16HBE cells was determined via western blotting and transmission electron microscopy. Necrosis assays of infected 16HBE cells were analyzed using propidium iodide staining and assessing lactate dehydrogenase (LDH) release. To determine how estradiol affects autophagy, infected 16HBE cells were treated with ER­specific and non­specific modulators. Reactive oxygen species (ROS) levels were analyzed via flow cytometry. Additionally, the protein expression levels of autophagy­associated proteins were determined via western blotting. Mtb could enter human lobar bronchial goblet cells and ciliated cells in patients with TBTB. The results also demonstrated that ERα was expressed in granulomatous tissue from patients with TBTB. Administration of 10­6 M estradiol reduced the number of intracellular Mtb colony­forming units in vitro in the 16HBE human bronchial epithelial cell line at day 3 after infection. Furthermore, cells treated with estradiol and infected with Mtb released less LDH at 72 h and exhibited reduced necrosis levels at 24 h compared with the untreated cells. In addition, autophagy of infected 16HBE cells was inhibited by estradiol. Estradiol and the specific ERα agonist had similar effects on autophagy in infected 16HBE cells. Additionally, treatment with the ERα antagonist abolished the inhibition of autophagy by estradiol in infected 16BHE cells. Compared with the untreated infected 16HBE cells, the ROS levels in the infected 16HBE cells treated with estradiol and the ERα agonist significantly decreased. The levels of phosphorylated (p)­mTOR and p­AKT notably increased in estradiol­ and ERα agonist­treated infected 16HBE cells. In summary, estradiol may serve a key role in the development of TBTB through binding to ERα.

One-pot synthesis of stable and functional hydrophilic CsPbBr3 perovskite quantum dots for "turn-on" fluorescence detection of Mycobacterium tuberculosis.

All-inorganic CsPbBr3 perovskite quantum dots (QDs) are widely studied owing to their excellent optoelectronic properties; however, they are usually hydrophobic and unstable in water and thus their biomedical applications are seriously limited. In this study, stable and hydrophilic CsPbBr3 QDs functionalized with carboxyl groups (CsPbBr3-COOH QDs) were prepared in one-pot with the aid of new ligands amino-poly(ethylene glycol)-carboxyl and perfluorooctyltriethoxylsilane. The aqueous solution of CsPbBr3-COOH QDs maintained the initial fluorescence intensity after 8 days of storage; the free carboxyl groups on the surface of CsPbBr3-COOH QDs were covalently conjugated with amino-terminal DNA to construct CsPbBr3 QDs-DNA probes for subsequent application. Then, a biosensing platform utilizing fluorescence resonance energy transfer between hydrophilic CsPbBr3 QDs-DNA and MoS2 nanosheets was developed for the sensitive and selective detection of the Mycobacterium tuberculosis DNA with a low limit of detection of 51.9 pM and the identification of drug-resistant clinical strains. This study advances the preparation of hydrophilic carboxyl-functionalized CsPbBr3 QDs with enhanced stability and extends their application in biomolecule detection.

Clinical characteristics and potential factors for recurrence of positive SARS-CoV-2 RNA in convalescent patients: a retrospective cohort study.

BACKGROUND: The recurrence of positive SARS-CoV-2 RT-PCR is frequently found in discharged COVID-19 patients but its clinical significance remains unclear. The potential cause, clinical characteristics and infectiousness of the recurrent positive RT-PCR patients need to be answered. METHODS: A single-centered, retrospective study of 51 discharged COVID-19 patients was carried out at a designated hospital for COVID-19. The demographic data, clinical records and laboratory findings of 25 patients with recurrent positive RT-PCR from hospitalization to follow-up were collected and compared to 26 patients with negative RT-PCR discharged regularly during the same period. Discharged patients' family members and close contacts were also interviewed by telephone to evaluate patients' potential infectiousness. RESULTS: The titer of both IgG and IgM antibodies was significantly lower (p = 0.027, p = 0.011) in patients with recurrent positive RT-PCR. Median duration of viral shedding significantly prolonged in patients with recurrent positive RT-PCR (36.0 days vs 9.0 days, p = 0.000). There was no significant difference in demographic features, clinical features, lymphocyte subsets count and inflammatory cytokines levels between the two groups of patients. No fatal case was noted in two groups. As of the last day of follow-up, none of the discharged patients' family members or close contact developed any symptoms of COVID-19. CONCLUSIONS: Patients with low levels of IgG and IgM are more likely to have recurrent positive SARS-CoV-2 RT-PCR results and lead to a prolonged viral shedding. The recurrent positive of SARS-CoV-2 RT-PCR may not indicate the recurrence or aggravation of COVID-19. The detection of SARS-CoV-2 by RT-PCR in the patients recovered from COVID-19 is not necessarily correlated with the ability of transmission.

Correlation between the variables collected at admission and progression to severe cases during hospitalization among patients with COVID-19 in Chongqing.

Mortality is high among severe patients with 2019 novel coronavirus-infected disease (COVID-19). Early prediction of progression to severe cases is needed. We retrospectively collected patients with COVID-19 in two hospital of Chongqing from 1st January to 29th February 2020. At admission, we collected the demographics and laboratory tests to predict whether the patient would progress to severe cases in hospitalization. Severe case was confirmed when one of the following criteria occurred: (a) dyspnea, respiratory rate ≥30 breaths/min, (b) blood oxygen saturation ≤93%, and (c) PaO2 /FiO2 ≤ 300 mm Hg. At admission, 348 mild cases were enrolled in this study. Of them, 20 (5.7%) patients progressed to severe cases after median 4.0 days (interquartile range: 2.3-6.0). Pulmonary inflammation index, platelet counts, sodium, C-reactive protein, prealbumin, and PaCO2 showed good distinguishing power to predict progression to severe cases (each area under the curve of receiver operating characteristics [AUC] ≥ 0.8). Age, heart rate, chlorine, alanine aminotransferase, aspartate aminotransferase, procalcitonin, creatine kinase, pH, CD3 counts, and CD4 counts showed moderate distinguishing power (each AUC between 0.7-0.8). And potassium, creatinine, temperature, and D-dimer showed mild distinguishing power (each AUC between 0.6-0.7). In addition, higher C-reactive protein was associated with shorter time to progress to severe cases (r = -0.62). Several easily obtained variables at admission are associated with progression to severe cases during hospitalization. These variables provide a reference for the medical staffs when they manage the patients with COVID-19.

Diagnostic value of rapid on-site evaluation during endobronchial ultrasound with a guide sheath for peripheral pulmonary lesions.

OBJECTIVE: To evaluate the applied value of rapid on-site evaluation during endobronchial ultrasound (EBUS) with a guide sheath for peripheral pulmonary lesions (PPLs). METHODS: Consecutive patients who underwent EBUS with a guide sheath for PPLs at our hospital from December 2015 to June 2017 in this retrospective study. The samples obtained from each operation were made rapid on-site evaluation at the same time. The results of rapid on-site evaluation were compared with the pathological diagnosis. RESULTS: A total of 127 PPLs in 124 patients were included in the study. 70 lesions were malignancy in the final pathological diagnosis. The sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy of rapid on-site evaluation for malignancy during EBUS with a guide sheath for PPLs was 88.6%, 98.2%, 98.4%, 87.5% and 92.9%, respectively. CONCLUSIONS: Rapid on-site evaluation during EBUS with a guide sheath has a high diagnostic value for malignant PPLs.

Immunocytochemistry Based on a Cell-Type-Specific Aptamer for Rapid Immunostaining of Adenocarcinoma Cells in Clinical Serosal Fluids.

All too often, conventional immunocytochemistry (ICC) via an antibody on cytological samples is limited to a few smears due to scant cellularity. To circumvent these limitations, this study employed a cell-type-specific aptamer as the core tool in ICC protocols for a timely and highly specific ICC diagnosis. S6, an aptamer against A549 lung carcinoma cells, was adopted instead of antibodies in this study for differentiating cancer cells in serosal fluids. Here, we developed three different strategies for discriminating the adenocarcinoma cells in effusion cytology specimens using the S6 aptamer in ICC. These strategies included a biotin-labeled S6 aptamer, an FAM-labeled S6 aptamer, and an activatable S6 aptamer. A total of 112 serosal fluid specimens with known diagnoses were evaluated by all three modes of use of the S6 aptamer. ICC procedures based on biotin-labeled or FAM-labeled S6 aptamers required time-consuming washing to avoid interference from nonspecific adsorption. ICC procedures based on an activatable S6 aptamer probe showed a weak fluorescence signal in the absence of target cells, but the procedures showed a strong fluorescence signal due to alteration of the conformation without any complicated washing steps, in the presence of targets. The specificity and sensitivity are higher in all three different ICC protocols based on the S6 aptamer than those for antibody protocols for differentiating adenocarcinoma cells in clinical effusion cytology. ICC based on cell-type-specific aptamers, instead of on a panel of a set of antibodies, is promising as an auxiliary method for the diagnosis of cancer.

[Value of C-ROSE During EBUS-TBNA to Obtain the Tissue Sample 
in the Diagnosis of Lung Cancer].

BACKGROUND: Most of the patients with lung and (or) mediastinal occupying lesions are considered to be primary lung cancer clinically, and endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a commonly useful operation to obtain the tissue sample and get definitive diagnosis of pathological tissues. In the EBUS-TBNA process, cytological rapid on-site evaluation (C-ROSE) is a useful technology. The purpose of our study is to discuss the value of C-ROSE in the diagnosis of lung cancer by EBUS-TBNA sampling. METHODS: Retrospective analysis of 141 cases clinical data who were performed with EBUS-TBNA and suspected diagnosis primary lung cancer, which were found have mediastinal and (or) lung lesions (including the enlargement of the lymph nodes/mass) by computed tomography (CT). Among these patients, 81 patients were in the C-ROSE group and 60 patients were in the No C-ROSE group. The message of puncture and complication of EBUS-TBNA with or without C-ROSE were compared. At the same time, we analysis the sensitivity and specificity, positive predictive value, negative predictive value of C-ROSE combined with EBUS-TBNA in that of the diagnosis of lung cancer. RESULTS: We found no statistical difference of the needle passes between C-ROSE group and No C-ROSE group. But in C-ROSE group, specimen qualified rate and diagnostic yields were signicantly higher than No C-ROSE group (98.77% vs 90.00%, 88.89% vs 75.00%, P<0.05), the incidence of complications in the C-ROSE group was signicantly lower than that in the No C-ROSE group (1.23% vs 11.67%, P<0.05). The sensitivity, specificity, positive predictive value and negative predictive value of C-ROSE combined with EBUS-TBNA in the diagnosis of lung cancer are 92.21%, 100.00%, 100.00% and 40.00%. CONCLUSIONS: EBUS-TBNA combined with C-ROSE can improve the specimen qualified rate and diagnostic rate, also can reduce the complications thus worthy of further promotion.