Efflux pump effects on levofloxacin resistance in Mycobacterium abscessus.

Mycobacterium abscessus (M. abscessus) inherently displays resistance to most antibiotics, with the underlying drug resistance mechanisms remaining largely unexplored. Efflux pump is believed to play an important role in mediating drug resistance. The current study examined the potential of efflux pump inhibitors to reverse levofloxacin (LFX) resistance in M. abscessus. The reference strain of M. abscessus (ATCC19977) and 60 clinical isolates, including 41 M. abscessus subsp. abscessus and 19 M. abscessus subsp. massilense, were investigated. The drug sensitivity of M. abscessus against LFX alone or in conjunction with efflux pump inhibitors, including verapamil (VP), reserpine (RSP), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), or dicyclohexylcarbodiimide (DCC), were determined by AlarmarBlue microplate assay. Drug-resistant regions of the gyrA and gyrB genes from the drug-resistant strains were sequenced. The transcription level of the efflux pump genes was monitored using qRT-PCR. All the tested strains were resistant to LFX. The drug-resistant regions from the gyrA and gyrB genes showed no mutation associated with LFX resistance. CCCP, DCC, VP, and RSP increased the susceptibility of 93.3% (56/60), 91.7% (55/60), 85% (51/60), and 83.3% (50/60) isolates to LFX by 2 to 32-fold, respectively. Elevated transcription of seven efflux pump genes was observed in isolates with a high reduction in LFX MIC values in the presence of efflux pump inhibitors. Efflux pump inhibitors can improve the antibacterial activity of LFX against M. abscessus in vitro. The overexpression of efflux-related genes in LFX-resistant isolates suggests that efflux pumps are associated with the development of LFX resistance in M. abscessus.

A metric for quantitative evaluation of glioma margin changes in magnetic resonance imaging.

BACKGROUND: Gliomas differ from meningiomas in their margins, most of which are not separated from the surrounding tissue by a distinct interface. PURPOSE: To characterize the margins of gliomas quantitatively based on the margin sharpness coefficient (MSC) is significant for clinical judgment and invasive analysis of gliomas. MATERIAL AND METHODS: The data for this study used magnetic resonance image (MRI) data from 67 local patients and 15 open patients to quantify the intensity of changes in the glioma margins of the brain using MSC. The accuracy of MSC was assessed by consistency analysis and Bland-Altman test analysis, as well as invasive correlations using receiver operating characteristic (ROC) and Spearman correlation coefficients for subjects. RESULTS: In grading the tumors, the mean MSC values were significantly lower for high-grade gliomas (HGG) than for low-grade gliomas (LGG). The concordance correlation between the measured gradient and the actual gradient was high (HGG: 0.981; LGG: 0.993), and the Bland-Altman mean difference at the 95% confidence interval (HGG: -0.576; LGG: 0.254) and the limits of concordance (HGG: 5.580; LGG: 5.436) indicated no statistical difference. The correlation between MSC and invasion based on the margins of gliomas showed an AUC of 0.903 and 0.911 for HGG and LGG, respectively. The mean Spearman correlation coefficient of the MSC versus the actual distance of invasion was -0.631 in gliomas. CONCLUSION: The relatively low MSC on the blurred margins and irregular shape of gliomas may help in benign-malignant differentiation and invasion prediction of gliomas and has potential application for clinical judgment.

STMP-Net: A Spatiotemporal Prediction Network Integrating Motion Perception.

This article proposes a video prediction network called STMP-Net that addresses the problem of the inability of Recurrent Neural Networks (RNNs) to fully extract spatiotemporal information and motion change features during video prediction. STMP-Net combines spatiotemporal memory and motion perception to make more accurate predictions. Firstly, a spatiotemporal attention fusion unit (STAFU) is proposed as the basic module of the prediction network, which learns and transfers spatiotemporal features in both horizontal and vertical directions based on spatiotemporal feature information and contextual attention mechanism. Additionally, a contextual attention mechanism is introduced in the hidden state to focus attention on more important details and improve the capture of detailed features, thus greatly reducing the computational load of the network. Secondly, a motion gradient highway unit (MGHU) is proposed by combining motion perception modules and adding them between adjacent layers, which can adaptively learn the important information of input features and fuse motion change features to significantly improve the predictive performance of the model. Finally, a high-speed channel is provided between layers to quickly transmit important features and alleviate the gradient vanishing problem caused by back-propagation. The experimental results show that compared with mainstream video prediction networks, the proposed method can achieve better prediction results in long-term video prediction, especially in motion scenes.

The aceE involves in mycolic acid synthesis and biofilm formation in Mycobacterium smegmatis.

BACKGROUND: The integrity of cell wall structure is highly significant for the in vivo survival of mycobacteria. We hypothesized that changes in morphology may indicate changes in cell wall metabolism and identified an aceE gene mutant (aceE-mut) which presented a deficient colony morphology on 7H10 agar by screening transposon mutagenesis in Mycolicibacterium smegmatis, basonym Mycobacterium smegmatis (M. smegmatis). This study aimed to identify the functional role of aceE gene in cell wall biosynthesis in M. smegmatis. RESULTS: We observed that the colony morphology of aceE-mut was quite different, smaller and smoother on the solid culture medium than the wild-type (WT) strain during the transposon library screening of M. smegmatis. Notably, in contrast with the WT, which aggregates and forms biofilm, the aceE-mut lost its ability of growing aggregately and biofilm formation, which are two very important features of mycobacteria. The morphological changes in the aceE-mut strain were further confirmed by electron microscopy which indicated smoother and thinner cell envelope images in contrast with the rough morphology of WT strains. Additionally, the aceE-mut was more fragile to acidic stress and exhibited a pronounced defects in entering the macrophages as compared to the WT. The analysis of mycolic acid (MA) using LC-MS indicated deficiency of alpha-MA and epoxy-MA in aceE-mut strain whereas complementation of the aceE-mut with a wild-type aceE gene restored the composition of MA. CONCLUSIONS: Over all, this study indicates that aceE gene plays a significant role in the mycolic acid synthesis and affects the colony morphology, biofilm formation of M. smegmatis and bacteria invasion of macrophage.

In vivo evolution of drug-resistant Mycobacterium tuberculosis in patients during long-term treatment.

BACKGROUND: In the current scenario, the drug-resistant tuberculosis is a significant challenge in the control of tuberculosis worldwide. In order to investigate the in vivo evolution of drug-resistant M. tuberculosis, the present study envisaged sequencing of the draft genomes of 18 serial isolates from four pre-extensively drug-resistant (pre-XDR) tuberculosis patients for continuous genetic alterations. RESULTS: All of the isolates harbored single nucleotide polymorphisms (SNPs) ranging from 1303 to 1309 with M. tuberculosis H37Rv as the reference. SNPs ranged from 0 to 12 within patients. The evolution rates were higher than the reported SNPs of 0.5 in the four patients. All the isolates exhibited mutations at sites of known drug targets, while some contained mutations in uncertain drug targets including folC, proZ, and pyrG. The compensatory substitutions for rescuing these deleterious mutations during evolution were only found in RpoC I491T in one patient. Many loci with microheterogeneity showed transient mutations in different isolates. Ninety three SNPs exhibited significant association with refractory pre-XDR TB isolates. CONCLUSIONS: Our results showed evolutionary changes in the serial genetic characteristics of the pre-XDR TB patients due to accumulation of the fixed drug-resistant related mutations, and the transient mutations under continuous antibiotics pressure over several years.

Utility of dominant epitopes derived from cell-wall protein LppZ for immunodiagnostic of pulmonary tuberculosis.

BACKGROUND: Serological antibodies tests for tuberculosis (TB) are widely used in developing countries. They appear to have some advantages- faster, simple and could be used for extrapulmonary TB. However, most of current commercial TB serological tests are failed to provide sufficient sensitivity and specificity. Improved serological biomarkers were essential. In this study, we present an approach using peptide array to discover new immunodiagnostic biomarkers based on immunodominant epitopes of TB antigens. RESULTS: The Probable conserved lipoprotein LppZ, which is difficult to express and purify in vivo was selected as the model antigen. We use two-step screening for dominant epitope selection. Based on peptide array data from 170 TB patients and 41 control samples, two dominant epitopes were identified to have diagnostic value for TB patients. Truncation assay was used to identify the core reactive sequence. Peptide- based ELISA was used to evaluate the diagnostic ability of pep-LppZ-1 and pep-LppZ-13. Pep-LppZ-1 has a sensitivity of 49.2% and a specificity of 83.3% in TB diagnose. Pep-LppZ-13 has a sensitivity of 43.3% and a specificity of 88.5% in TB diagnose. CONCLUSIONS: Our result demonstrated that peptide array screening would be an advantage strategy of screening TB diagnostic peptides.

Precision methylome characterization of Mycobacterium tuberculosis complex (MTBC) using PacBio single-molecule real-time (SMRT) technology.

Tuberculosis (TB) remains one of the most common infectious diseases caused by Mycobacterium tuberculosis complex (MTBC). To panoramically analyze MTBC's genomic methylation, we completed the genomes of 12 MTBC strains (Mycobacterium bovis; M. bovis BCG; M. microti; M. africanum; M. tuberculosis H37Rv; H37Ra; and 6 M. tuberculosis clinical isolates) belonging to different lineages and characterized their methylomes using single-molecule real-time (SMRT) technology. We identified three (m6)A sequence motifs and their corresponding methyltransferase (MTase) genes, including the reported mamA, hsdM and a newly discovered mamB. We also experimentally verified the methylated motifs and functions of HsdM and MamB. Our analysis indicated the MTase activities varied between 12 strains due to mutations/deletions. Furthermore, through measuring 'the methylated-motif-site ratio' and 'the methylated-read ratio', we explored the methylation status of each modified site and sequence-read to obtain the 'precision methylome' of the MTBC strains, which enabled intricate analysis of MTase activity at whole-genome scale. Most unmodified sites overlapped with transcription-factor binding-regions, which might protect these sites from methylation. Overall, our findings show enormous potential for the SMRT platform to investigate the precise character of methylome, and significantly enhance our understanding of the function of DNA MTase.

Wild-Type and Non-Wild-Type Mycobacterium tuberculosis MIC Distributions for the Novel Fluoroquinolone Antofloxacin Compared with Those for Ofloxacin, Levofloxacin, and Moxifloxacin.

Antofloxacin (AFX) is a novel fluoroquinolone that has been approved in China for the treatment of infections caused by a variety of bacterial species. We investigated whether it could be repurposed for the treatment of tuberculosis by studying its in vitro activity. We determined the wild-type and non-wild-type MIC ranges for AFX as well as ofloxacin (OFX), levofloxacin (LFX), and moxifloxacin (MFX), using the microplate alamarBlue assay, of 126 clinical Mycobacterium tuberculosis strains from Beijing, China, of which 48 were OFX resistant on the basis of drug susceptibility testing on Löwenstein-Jensen medium. The MIC distributions were correlated with mutations in the quinolone resistance-determining regions of gyrA (Rv0006) and gyrB (Rv0005). Pharmacokinetic/pharmacodynamic (PK/PD) data for AFX were retrieved from the literature. AFX showed lower MIC levels than OFX but higher MIC levels than LFX and MFX on the basis of the tentative epidemiological cutoff values (ECOFFs) determined in this study. All strains with non-wild-type MICs for AFX harbored known resistance mutations that also resulted in non-wild-type MICs for LFX and MFX. Moreover, our data suggested that the current critical concentration of OFX for Löwenstein-Jensen medium that was recently revised by the World Health Organization might be too high, resulting in the misclassification of phenotypically non-wild-type strains with known resistance mutations as wild type. On the basis of our exploratory PK/PD calculations, the current dose of AFX is unlikely to be optimal for the treatment of tuberculosis, but higher doses could be effective.

A subunit vaccine based on rH-NS induces protection against Mycobacterium tuberculosis infection by inducing the Th1 immune response and activating macrophages.

Mycobacterium tuberculosis (Mtb) is a Gram-positive pathogen which causes tuberculosis in both animals and humans. All tested rH-NS formulations induced a specific Th1 response, as indicated by increased production of interferon γ (IFN-γ) and interleukin 2 (IL-2) by lymphocytes in the spleen of mice which were immunized with rH-NS alone or with rH-NS and the adjuvant cyclic GMP-AMP (cGAMP). Serum from mice immunized with rH-NS with or without adjuvant also had higher levels of IL-12p40 and TNF-α, compared with those from control mice immunized with phosphate-buffered saline. Both vaccines increased protective efficacy in mice which were challenged with Mtb H37Rv, as measured by reduced relative CFU counts in the lungs. We found that rH-NS induced the production of TNF-α, IL-6, and IL-12p40, which relied on the activation of mitogen-activated protein kinases by stimulating the rapid phosphorylation of ERK1/2, p38, and JNK, and on the activation of transcription factor NF-κB in macrophages. Additionally, we also found that rH-NS could interact with TLR2 but not TLR4 in pull-down assays. The rH-NS-induced cytokine production from TLR2-silenced RAW264.7 cells was lower than that from BALB/c macrophages. Prolonged exposure (>24 h) of RAW264.7 cells to rH-NS resulted in a significant enhancement in IFN-γ-induced MHC II expression, which was not found in shTLR2-treated RAW264.7 cells. These results suggest that rH-NS is a TLR2 agonist which induces the production of cytokines by macrophages and up-regulates macrophage function.

Bacteriological and virulence study of a Mycobacterium chimaera isolate from a patient in China.

A clinical isolate from a patient was identified as Mycobacterium chimaera, a recently identified species of nontuberculous Mycobacteria. The biochemical and molecular identity, drug sensitivity and virulence of this isolated strain were investigated. 16S rRNA, the 16S-23S ITS, hsp65 and rpoB were amplified, and their sequence similarities with other mycobacteria were analyzed. The minimum inhibitory concentrations of 22 anti-microbial agents against this isolate were established, and the virulence of the isolate was evaluated by intravenous injection into C57BL/6 mice using Mycobacterium tuberculosis H37Rv as a control strain. Growth and morphological characteristics and mycolic acid profile analysis revealed that this isolated strain was a member of the Mycobacterium avium complex. BLAST analysis of the amplified sequences showed that the isolated strain was closely related to M. chimaera. Susceptibility testing showed that the isolate was sensitive to rifabutin, rifapentine, clarithromycin, azithromycin, imipenem and cefoxitin. Bacterial load determination and tissue histopathology of the infected mice indicated that the isolate was highly virulent. The first case of M. chimaera infection in China was evaluated. The information derived from this case may offer valuable guidance for clinical diagnosis and treatment.

[Detection of mutations in the gyrA and gyrB genes associated with fluoroquinolone-resistance among clinical isolates of Mycobacterium abscessus in China].

OBJECTIVE: To determine molecular mutations in gyrA and gyrB genes and their association with fluoroquinone-resistance among Mycobacterium abscessus isolates from China. METHODS: Antimicrobial susceptibility profile of Mycobacterium abscessus isolates was evaluated for resistance to levofloxacin (LFX) and moxifloxacin (MFX). The quinolone resistant determing regions (QRDR) of gyrA and gyrB genes of all the isolates was sequenced and analyzed. RESULTS: A total 70 Mycobacterium abscessus isolates were analyzed, including 45 (64%) M. abscessus subsp. abscessus and 25 (36%) M. abscessus subsp. massiliense. Analysis of antimicrobial susceptibility profile showed 97% (n=68) of isolates were resistance to LFX and 14.3% (n=10) of isolates were resistance to MFX. All the MFX-resistant strains were resistant to LFX. Cross-resistance to LFX and MFX was noted in 14.3% (n=10) of isolates. Sequencing analysis revealed that the Ser83Ala substitution in the gyrA gene and Lys447Arg and Ser464Asn substitutions in the gyrB gene were found in all the isolates from both M. abscessus subsp. abscessus and M. abscessus subsp. massiliense. No mutation was found to be associated with cross-resistance of M. abscessus to LFX and MFX in the entire gyrA and gyrB gene. CONCLUSIONS: Our data suggest that there is no clear correlation between the type of mutation in the gene gyrA and gyrB, and the MIC levels of LFX and MFX for resistant M. abscessus strains.

Recombinant MPT83 derived from Mycobacterium tuberculosis induces cytokine production and upregulates the function of mouse macrophages through TLR2.

TLR2 recognizes components of Mycobacterium tuberculosis and initiates APC activities that influence both innate and adaptive immunity. M. tuberculosis lipoproteins are an important class of TLR2 ligands. In this study, we focused on recombinant MPT83 (rMPT83) to determine its effects on mouse macrophages. We demonstrated that rMPT83 induced the production of TNF-α, IL-6, and IL-12 p40 and that cytokine induction depended on activated MAPKs, because we observed the rapid phosphorylation of ERK1/2, p38, and JNK in macrophages. Additionally, neutralizing Abs against TLR2 significantly inhibited cytokine secretion and reduced or attenuated the rMPT83-induced activation of p38 and JNK in RAW264.7 cells, a mouse macrophage cell line. Furthermore, rMPT83-induced cytokine production was significantly lower in macrophages from TLR2(-/-) mice than in macrophages from wild-type mice. We further found that prolonged exposure (>24 h) of RAW264.7 cells or macrophages from wild-type and TLR2(-/-) mice to rMPT83 resulted in a significant enhancement of IFN-γ-induced MHC class II expression and an enhanced ability of macrophages to present the rMPT83 peptide to CD4(+) T cells. These results indicated that rMPT83 is a TLR2 agonist that induces the production of cytokines by macrophages and upregulates macrophage function.

Medical and nursing students' satisfaction with e-learning platforms during the COVID-19 pandemic: Initial findings of an experimental project in China.

Satisfaction with learning management systems (LMSs) is an essential indicator of students' e-learning experiences and reflects the quality of e-learning. Applying the technology satisfaction model, the present study aimed to investigate medical and nursing students' satisfaction with LMSs and its predictors. We conducted our survey at a medical university located in East China and received a total of 329 effective responses. Structural equation modelling was used to analyse the data. Our findings confirmed that perceived ease of use and perceived usefulness were two direct predictors of medical and nursing students' satisfaction with LMSs. Furthermore, the influence of perceived usefulness on satisfaction was more powerful than that of perceived ease of use. This study also substantiated that computer self-efficacy and perceived ease of use can indirectly impact medical and nursing students' satisfaction with LMSs. Our research effectively links the theoretical hypotheses with empirical findings, highlighting the central role of Computer Self-Efficacy (CSE), perceived ease of use, and perceived usefulness in shaping medical and nursing students' satisfaction with LMSs. Our findings contributed to the understanding of the technology satisfaction model and medical and nursing students' e-learning during the COVID-19 pandemic.

Efficacy of clofazimine-containing regimens for treatment of Mycobacterium avium complex-pulmonary disease in patients unsuitable for standard treatment regimen.

OBJECTIVES: Patients with Mycobacterium avium complex-pulmonary disease (MAC-PD) can exhibit contraindications in applying the recommended treatment regimens by the guidelines. Clofazimine (CFZ) is considered a promising drug for MAC-PD treatment and is frequently included in alternative regimens; however, its efficacy remains unclear. METHODS: MAC-PD patients, unsuitable for standard regimens, were enrolled continuously in a prospective study at Beijing Chest Hospital. The treatment response of the CFZ-containing regimen was monitored. RESULTS: Fifty patients were enrolled in the initial treatment, and 25 patients had a history of anti-TB treatment. Nodular bronchiectasis was observed in 34 patients, while 8 patients exhibited fibrocavitary changes. Additionally, eight patients displayed a combination of both patterns. In a multivariate analysis, MAC-PD patients with CFZ MIC < 0.25 mg/L were significantly associated with culture conversion [OR 8.415, 95% CI (1.983-35.705); P = 0.004]. Among patients who had previous TB treatment history, patients with CFZ MIC < 0.25 mg/L had a higher chance of acquiring culture conversion outcomes [(OR 7.737, 95% CI 1.032-57.989); P = 0.046]. In contrast, among patients with no previous TB treatment history, the RIF-containing regimen had a higher chance of acquiring culture conversion outcomes [(OR 11.038, 95%CI 1.008-120.888); P = 0.049]. CONCLUSION: MAC-PD patients unsuitable for standard regimens could benefit from a CFZ-containing regimen, especially for patients with previous TB treatment history and baseline CFZ MIC values lower than 0.25 mg/L.

Tuberculosis-targeted next-generation sequencing and machine learning: An ultrasensitive diagnostic strategy for paucibacillary pulmonary tuberculosis and tuberculous meningitis.

BACKGROUND: Existing diagnostic approaches for paucibacillary tuberculosis (TB) are limited by the low sensitivity of testing methods and difficulty in obtaining suitable samples. METHODS: An ultrasensitive TB diagnostic strategy was established, integrating efficient and specific TB targeted next-generation sequencing and machine learning models, and validated in clinical cohorts to test plasma cfDNA, cerebrospinal fluid (CSF) DNA collected from tuberculous meningitis (TBM) and pediatric pulmonary TB (PPTB) patients. RESULTS: In the detection of 227 samples, application of the specific thresholds of CSF DNA (AUC = 0.974) and plasma cfDNA (AUC = 0.908) yielded sensitivity of 97.01 % and the specificity of 95.65 % in CSF samples and sensitivity of 82.61 % and specificity of 86.36 % in plasma samples, respectively. In the analysis of 44 paired samples from TBM patients, our strategy had a high concordance of 90.91 % (40/44) in plasma cfDNA and CSF DNA with both sensitivity of 95.45 % (42/44). In the PPTB patient, the sensitivity of the TB diagnostic strategy yielded higher sensitivity on plasma specimen than Xpert assay on gastric lavage (28.57 % VS. 15.38 %). CONCLUSIONS: Our TB diagnostic strategy provides greater detection sensitivity for paucibacillary TB, while plasma cfDNA as an easily collected specimen, could be an appropriate sample type for PTB and TBM diagnosis.

Bedaquiline resistance pattern in clofazimine-resistant clinical isolates of tuberculosis patients.

OBJECTIVES: Bedaquiline (BDQ) is a potent drug for treating drug-resistant tuberculosis (TB). Here, we analysed the resistance profiles of BDQ in CFZ-resistant clinical isolates and investigated the clinical risk factors of BDQ and CFZ cross/co-resistance. METHODS: The AlarmarBlue microplate assay was performed to determine the minimum inhibitory concentration (MIC) of the CFZ-resistant Mycobacterium tuberculosis (MTB) clinical isolates to CFZ and BDQ. The clinical characteristics of the respective patients were analysed to explore the possible risk factors of BDQ resistance. The drug-resistance-associated genes including Rv0678, Rv1979c, atpE, pepQ and Rv1453 were sequenced and analysed. RESULTS: A total of 72 clinical CFZ-resistant MTB isolates were collected; among these, half were identified as BDQ-resistant. The MIC value of BDQ closely correlated with CFZ (Spearman's q = 0.766, P < 0.005). Among the isolates with a MIC of CFZ ≥4 mg/L, 92.31% (12/13) were resistant to BDQ. Pre-XDR and exposure to BDQ or CFZ are the major risk factors for concurrent BDQ resistance. Among the 36 cross/co-resistant isolates, 50% (18/36) had mutations in Rv0678, 8.3% (3/36) had mutations in Rv0678+Rv1453, 5.6% (2/36) had mutations in Rv0678+Rv1979c, 2.8% (1/36) had mutations in Rv0678+Rv1979c+Rv1453, 2.8% (1/36) had mutations in atpE+Rv0678+Rv1453, 2.8% (1/36) had mutations in Rv1979c, and 27.7% (10/36) had no variations in the target genes. CONCLUSION: Nearly half of the CFZ-resistant isolates were still sensitive to BDQ, whereas this rate dramatically decreased among patients with pre-XDR TB or those who had been exposed to BDQ or CFZ.

Oral immunogenicity of potato-derived antigens to Mycobacterium tuberculosis in mice.

The novel use of transgenic plants as vectors for the expression of viral and bacterial antigens has been increasingly tested as an alternative methodology for the production and delivery of experimental oral vaccines. Here, we examined the immunogenicity of combined plant-made vaccines that include four genes encoding immune-dominant antigens from Mycobacterium tuberculosis. Compared with the wild type and other control groups, mice treated with the combined plant-made vaccines showed significantly higher levels of interferon-γ and interleukin-2 production in response to all four proteins, and higher levels of antigen-specific CD4(+) and CD8(+) T-cell responses and immunoglobulin (Ig) G and IgA titers. These results suggest that combined plant-made vaccines can induce immunogenicity against M. tuberculosis through the induction of stronger Th1-associated immune responses. This is the first report of an orally delivered combined plant-made vaccine against tuberculosis priming an antigen-specific Th1 response, a comprehensive effect including both mucosal and systemic immune responses.

Differential Acceptance of Advance Directives Between Millennials and Baby Boomer Generations: A Cross-Sectional Survey Study Among College Students and Their Relatives.

Objectives: This study aims to explore the differentials of knowledge and attitude of advance directives (ADs) between millennials and baby boomer generations, and the effects of the intention to sign the advance directives. Methods: This is a cross-sectional study using a self-administered questionnaire to collect data from 325 students in a health-related college of a University of Science and Technology in Taiwan, and their parents, as total of 226, who are baby boomers. The statistical methods include descriptive statistics and inferential statistics. Results: Only 10 people from the 2 generations signed an AD. The multivariate logistic regression showed that baby boomer generation, AD knowledge, and AD attitude were significant positive associate of willingness to sign AD in the future. Conclusions: The government may enhance promotion of ADs among millennials and improve the connection between millennials' knowledge of and attitude toward ADs, and their AD signing behavior.

Prevalence and related factors of depressive symptoms among HIV/AIDS in Ningbo, China: A cross-sectional study.

Background: Depressive symptoms were common among HIV/AIDS patients. Previous studies had shown that HIV-infected patients were twice as likely to be diagnosed with depression as the general population. However, only few studies have explored the prevalence and related factors of depressive symptoms among HIV/AIDS in China. Materials and methods: A cross-sectional study was conducted to study the prevalence of depressive symptoms among HIV/AIDS from January to December 2021 through the database of HIV/AIDS antiretroviral therapy and psychological evaluation system in Ningbo, China. The Patient Health Questionnaire-2 (PHQ-2) was used to screen for depressive symptoms (PHQ-2 > 0), the Patient Health Questionnaire-9 (PHQ-9) was used to diagnose depressive symptoms, and multivariate Logistic regression model was carried on to evaluate the related factors. Results: A total of 3,939 HIV/AIDS patients were enrolled, and the age of initiation of antiretroviral therapy was 37.15 (IQR = 28.41-48.73) years. Among them, 3,230 (82.00%) were male, 3,844 (97.59%) were Han nationality, 1,391 (35.49%) were unmarried, 1,665 (42.27%) were homosexual transmission, and 2,194 (55.70%) were HIV-infected patients. There were 265 patients (6.73%) with depressive symptoms, and the proportion of mild, moderate, moderate and severe depressive symptoms was 4.01% (158/3939), 1.65% (65/3939), 0.76% (30/3939), and 0.30% (12/3939), respectively. Multivariate analysis showed that married [odds ratio (OR) = 0.675, 95% CI = 0.501-0.908], divorced or widowed (OR = 0.571, 95% CI = 0.380-0.860), homosexual transmission (OR = 1.793, 95% CI = 1.349-2.396) were associated with depressive symptoms among HIV/AIDS. Conclusion: The prevalence of depressive symptoms among HIV/AIDS patients was 6.73% in Ningbo, China. More attention should be paid to the psychological status of unmarried and homosexual HIV/AIDS patients in Ningbo and timely psychological intervention or treatment should be given to those patients with depressive symptoms.

Doubled Nontuberculous Mycobacteria Isolation as a Consequence of Changes in the Diagnosis Algorithm.

Background: Nontuberculous mycobacteria (NTM) and their associated diseases remain neglected. Through minor modifications in our diagnostic algorithm, we observed an unexpected higher number of cultivable NTM isolates. Therefore, a retrospective study was performed thoroughly to investigate the effect of changed laboratory procedures on NTM isolation in a specialized tuberculosis hospital. Methods: NTM isolation rates and composition of NTM species were compared for the two diagnostic algorithms: (1) by using traditional p-nitrobenzoic acid (PNB) selective medium as a preliminary test to identify NTM isolates among the positive cultures (procedure I) and (2) by using the MPT64 antigen detection method to distinguish between Mycobacterium tuberculosis complex (MTBC) isolates and possible NTM isolates after a positive MGIT960 liquid culture (procedure II). Results: The NTM isolation rate in procedure II was significantly higher than the procedure I (18.08% vs 9.71%; P<0.001). A noticeable increase in the ratio of NTM isolates among the identified mycobacteria was observed over the studied years (ie, from 58.18% in 2019 to 72.93% in 2021), which indicated a more precise prescription of species identification test after prompt information was provided in procedure II. In addition, the consistency of the identified species using multiple specimens from the same patient did not present a significant difference between the procedures. Conclusion: According to our study, NTM infection might be far more underestimated than it is. A diagnostic procedure combining MGIT960 culture and MPT64 antigen detection could timely and easily identify clues of NTM isolates and improve the diagnosis of NTM infections.