ABSTRACT
BACKGROUND: The GeneXpert MTB/RIF (Xpert) assay is a widely used technology for detecting Mycobacterium tuberculosis (MTB) in clinical samples. However, the study on the failure of the Xpert assay during routine implementation and its potential solutions is limited. METHODS: We retrospectively analyzed the records of unsuccessful tests in the Xpert and the GeneXpert MTB/RIF Ultra (Ultra) assays between April 2017 and April 2021 at the Shanghai Public Health Clinical Center. To further investigate the effect of prolonged preprocessing on clinical sputum, an additional 120 sputum samples were collected for Xpert testing after 15 min, 3 h, and 6 h preprocessing. The analysis was performed by SPSS version 19.0 software. RESULTS: A total of 11,314 test records were analyzed, of which 268 (2.37%) had unsuccessful test results. Among these, 221 (1.95%) were reported as "Error", 43 (0.38%) as "Invalid", and 4 (0.04%) as "No result". The most common clinical specimen for Xpert tests was sputum, accounting for 114 (2.17%) unsuccessful tests. The failure rate of urine specimens was lower than that of sputum (OR = 0.12, 95% CI: 0.02-0.88, χ2 = 6.22, p = 0.021). In contrast, the failure rate of stool specimens was approximately twice as high as that of sputum (OR = 1.93, 95% CI: 1.09-3.40, χ2 = 5.35, p = 0.014). In the prolonged preprocessing experiment, 102 cases (85%) yielded consistent results in Xpert tests. Furthermore, 7 cases (5.83%) detected an increase in MTB load, 8 cases (6.67%) detected a decrease in MTB load, and 3 cases (2.5%) yielded incongruent results in MTB and rifampicin resistance detection. CONCLUSIONS: The primary cause of unsuccessful tests in the Xpert assay was reported as "Error". Despite varying failure rates depending on the samples, the Xpert assay can be applied to extrapulmonary samples. For paucibacillary specimens, retesting the remaining preprocessed mixture should be carefully considered.
Subject(s)
Mycobacterium tuberculosis , Sputum , Humans , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/isolation & purification , Sputum/microbiology , Retrospective Studies , China , Specimen Handling/methods , Molecular Diagnostic Techniques/methods , Tuberculosis/diagnosis , Tuberculosis/microbiology , Rifampin/pharmacology , Tuberculosis, Pulmonary/diagnosis , Tuberculosis, Pulmonary/microbiology , Male , FemaleABSTRACT
Trends in and risk factors for drug resistance in Mycobacterium tuberculosis (M. tuberculosis) in human immunodeficiency virus (HIV)-infected patients with active tuberculosis were analyzed. The clinical data of M. tuberculosis and HIV-coinfected patients treated at the Shanghai Public Health Clinical Center between 2010 and 2022 were collected. The diagnosis of tuberculosis was confirmed by solid or liquid culture. The phenotypic drug susceptibility test was carried out via the proportional method, and the resistance to first-line and second-line drugs was analyzed. Logistic regression analysis was performed to identify associated risk factors for drug resistance in M. tuberculosis. Of the 304 patients with a M. tuberculosis-positive culture and first-line drug susceptibility test results, 114 (37.5%) were resistant to at least one first-line anti-tuberculosis drug. Of the 93 patients with first-line and second-line drug susceptibility test results, 40 (43%) were resistant to at least one anti-tuberculosis drug, and 20 (21.5%), 27 (29.0%), 19 (20.4%), 16 (17.2%), and 14 (15.1%) were resistant to rifampicin, streptomycin, ofloxacin, levofloxacin, and moxifloxacin, respectively; 17 patients (18.3%) had multidrug-resistant tuberculosis (MDR-TB). Between 2010 and 2021, the rate of resistance to streptomycin and rifampicin ranged from 14.3% to 40.0% and from 8.0% to 26.3%, respectively, showing an increasing trend year by year. From 2016 to 2021, the rate of resistance to quinolones fluctuated between 7.7% and 27.8%, exhibiting an overall upward trend. Logistic regression analysis showed that being aged <60 years old was a risk factor for streptomycin resistance, mono-drug resistance, and any-drug resistance (RR 4.139, p = 0.023; RR 7.734, p = 0.047; RR 3.733, p = 0.009). Retreatment tuberculosis was a risk factor for resistance to rifampicin, ofloxacin, of levofloxacin (RR 2.984, p = 0.047; RR 4.517, p = 0.038; RR 6.277, p = 0.014). The drug resistance rates of M. tuberculosis to rifampicin and to quinolones in HIV/AIDS patients were high and have been increasing year by year. Age and a history of previous anti-tuberculosis treatment were the main factors associated with the development of drug resistance in HIV/AIDS patients with tuberculosis.
Subject(s)
Antitubercular Agents , HIV Infections , Microbial Sensitivity Tests , Mycobacterium tuberculosis , Tuberculosis, Multidrug-Resistant , Humans , Mycobacterium tuberculosis/drug effects , Risk Factors , Female , Male , HIV Infections/complications , HIV Infections/microbiology , HIV Infections/drug therapy , Adult , Antitubercular Agents/pharmacology , Antitubercular Agents/therapeutic use , Middle Aged , Tuberculosis, Multidrug-Resistant/microbiology , Tuberculosis, Multidrug-Resistant/drug therapy , Tuberculosis, Multidrug-Resistant/epidemiology , China/epidemiology , Coinfection/microbiology , Coinfection/drug therapy , Drug Resistance, Multiple, Bacterial , Young Adult , Drug Resistance, Bacterial , AgedABSTRACT
BACKGROUND: The pharmacokinetic (PK) and pharmacodynamic (PD) characteristics of nirmatrelvir (NMV) are unknown in Chinese patients with COVID-19. OBJECTIVES: To understand the PK, as well as PK-PD characteristics of NMV for optimizing the dose in Chinese patients with COVID-19. METHODS: We enrolled 141 participants who received NMV 300 mg/ritonavir (RTV) 100 mg b.i.d. for 5 days. The NMV concentrations were analyzed using 251 blood samples. PK/PD of NMV was investigated in these COVID-19 patients using a nonlinear mixed-effects model. RESULTS: The patients had a mean age of 82 years (range, 34-97). The absorption rate constant and apparent clearance of NMV in this Chinese cohort were 0.253 h-1 and 6.83 L/h, respectively, similar to Caucasian patients. No covariates affected NMV clearance. Predicted peak (Cmax) and trough concentration (Cmin) under 300 mg NMV/100 mg RTV b.i.d. were 4004 and 1498 ng/mL, respectively. Although higher AUC and Cmin were weakly associated with a slight increase in the number of cycle threshold (CT) of viral genes, no significant correlation was found, indicating a weak relationship between drug exposure and efficacy (CT). CONCLUSIONS: In all, our findings suggest no ethnic PK differences, a weak and clinically insignificant relationship between drug exposure and efficacy, suitable dosage for Chinese patients (including the elderly) based on PK parameters, and the need for further studies to determine optimal regimens for high-risk patients due to inter-individual variability.
Subject(s)
COVID-19 Drug Treatment , Ritonavir , Humans , Male , Aged , Middle Aged , Female , Aged, 80 and over , Ritonavir/pharmacokinetics , Ritonavir/therapeutic use , Ritonavir/administration & dosage , Ritonavir/pharmacology , Adult , Antiviral Agents/pharmacokinetics , Antiviral Agents/pharmacology , Antiviral Agents/administration & dosage , Asian People , SARS-CoV-2 , China , COVID-19 , East Asian PeopleABSTRACT
T cell/B cell mixed phenotypic lymphocytes have been observed in different disease contexts, yet their presence and function in physiological conditions remain elusive. Here, we provide evidence for the existence of a lymphocyte subset endogenously expressing both T- and B-cell lineage markers in mice. The majority of these T/B phenotypic lymphocytes (CD3+CD19+) show an origin of pro/pre B cells and distribute widely in mouse bone marrow, lymph nodes, spleen, and peripheral blood. Functional assays show that these biphenotypic lymphocytes can be activated through stimulating TCR or BCR signaling pathways. Moreover, we show that these cells actively participate both the humoral and cellular immune responses elicited by vaccination. Compared to conventional T cells, these biphenotypic lymphocytes can secrete a higher level of IL-2 but a lower level of TNF-α upon antigen specific stimulation. An equivalent lymphocyte subset is found in freshly isolated human PBMCs and exhibits similar functionality, albeit at a lower frequency than in mice.
Subject(s)
B-Lymphocytes , Lymphocyte Subsets , Humans , Animals , Mice , Adaptor Proteins, Signal Transducing , Biological Assay , Lymph NodesABSTRACT
Objectives: The aim of this study was to evaluate the performance of a new double-antigen sandwich test that is based on the light-initiated chemiluminescent assay (LiCA®) for detecting anti-hepatitis C virus antibodies (anti-HCV) in comparison to Architect®. Methods: Analytical characteristics and diagnostic performance were tested using seroconversion panels and large pools of clinical samples. Positive results were validated by the strip immunoblot assay (RIBA) and HCV RNA. Results: Repeatability and within-lab imprecision of LiCA® anti-HCV were 1.31%-3.27%. The C5-C95 interval was -5.44%-5.03% away from C50. LiCA® detected seroconversion in an average of 28.9 days and showed a mean of 3.7 (p = 0.0056) days earlier than Architect®. In a pool of 239 samples with known HCV genotypes 1 to 6, both assays correctly detected all subjects. In 16,305 clinical patient sera, LiCA® detected 4 false-negative (0.25) and 14 false-positive (0.86) anti-HCV cases, while Architect® recorded 6 false-negative (0.37) and 138 false-positive (8.46) subjects, respectively. Compared to Architect®, LiCA® presented a significantly better performance in specificity (99.91% vs. 99.14%, n = 16,018, p < 0.0001), positive predictive value (95.29% vs. 67.06%, n = 419, p < 0.0001), and overall accuracy (99.89% vs. 99.12%, n = 16,305, p < 0.0001), while no significant difference in sensitivity (98.61% vs. 97.91%, n = 287, p = 0.5217) and negative predictive value (99.98% vs. 99.96%, n = 15,886, p = 0.3021) was seen. An S/Co value of 3.28 was predicted to be the threshold with a positivity ≥95% for the LiCA® anti-HCV assay. Conclusion: LiCA® anti-HCV is a precise and fully automatic chemiluminescent assay with superior sensitivity and specificity. The assay can be used as a valuable tool to supplement the diagnosis of HCV infection.
Subject(s)
Hepatitis C Antibodies , Hepatitis C , Humans , Luminescent Measurements/methods , Sensitivity and Specificity , RNA, Viral/genetics , Hepatitis C/diagnosisSubject(s)
COVID-19 , Coinfection , Humans , Coinfection/epidemiology , Pandemics , China/epidemiologyABSTRACT
Coronavirus disease 2019 (COVID-19) severity has been associated with alterations of the gut microbiota. However, the relationship between gut microbiome alterations and COVID-19 prognosis remains elusive. Here, we performed a genome-resolved metagenomic analysis on fecal samples from 300 in-hospital COVID-19 patients, collected at the time of admission. Among the 2,568 high quality metagenome-assembled genomes (HQMAGs), redundancy analysis identified 33 HQMAGs which showed differential distribution among mild, moderate, and severe/critical severity groups. Co-abundance network analysis determined that the 33 HQMAGs were organized as two competing guilds. Guild 1 harbored more genes for short-chain fatty acid biosynthesis, and fewer genes for virulence and antibiotic resistance, compared with Guild 2. Based on average abundance difference between the two guilds, the guild-level microbiome index (GMI) classified patients from different severity groups (average AUROC [area under the receiver operating curve] = 0.83). Moreover, age-adjusted partial Spearman's correlation showed that GMIs at admission were correlated with 8 clinical parameters, which are predictors for COVID-19 prognosis, on day 7 in hospital. In addition, GMI at admission was associated with death/discharge outcome of the critical patients. We further validated that GMI was able to consistently classify patients with different COVID-19 symptom severities in different countries and differentiated COVID-19 patients from healthy subjects and pneumonia controls in four independent data sets. Thus, this genome-based guild-level signature may facilitate early identification of hospitalized COVID-19 patients with high risk of more severe outcomes at time of admission. IMPORTANCE Previous reports on the associations between COVID-19 and gut microbiome have been constrained by taxonomic-level analysis and overlook the interaction between microbes. By applying a genome-resolved, reference-free, guild-based metagenomic analysis, we demonstrated that the relationship between gut microbiota and COVID-19 is genome-specific instead of taxon-specific or even species-specific. Moreover, the COVID-19-associated genomes were not independent but formed two competing guilds, with Guild 1 potentially beneficial and Guild 2 potentially more detrimental to the host based on comparative genomic analysis. The dominance of Guild 2 over Guild 1 at time of admission was associated with hospitalized COVID-19 patients at high risk for more severe outcomes. Moreover, the guild-level microbiome signature is not only correlated with the symptom severity of COVID-19 patients, but also differentiates COVID-19 patients from pneumonia controls and healthy subjects across different studies. Here, we showed the possibility of using genome-resolved and guild-level microbiome signatures to identify hospitalized COVID-19 patients with a high risk of more severe outcomes at the time of admission.
Subject(s)
COVID-19 , Gastrointestinal Microbiome , Microbiota , Humans , Gastrointestinal Microbiome/genetics , Feces , PrognosisABSTRACT
BACKGROUND: Viral persistence is a crucial factor that influences the transmissibility of SARS-CoV-2. However, the impacts of vaccination and physiological variables on viral persistence have not been adequately clarified. METHODS: We collected the clinical records of 377 COVID-19 patients, which contained unvaccinated patients and patients received two doses of an inactivated vaccine or an mRNA vaccine. The impacts of vaccination on disease severity and viral persistence and the correlations between 49 laboratory variables and viral persistence were analyzed separately. Finally, we established a multivariate regression model to predict the persistence of viral RNA. RESULTS: Both inactivated and mRNA vaccines significantly reduced the rate of moderate cases, while the vaccine related shortening of viral RNA persistence was only observed in moderate patients. Correlation analysis showed that 10 significant laboratory variables were shared by the unvaccinated mild patients and mild patients inoculated with an inactivated vaccine, but not by the mild patients inoculated with an mRNA vaccine. A multivariate regression model established based on the variables correlating with viral persistence in unvaccinated mild patients could predict the persistence of viral RNA for all patients except three moderate patients inoculated with an mRNA vaccine. CONCLUSION: Vaccination contributed limitedly to the clearance of viral RNA in COVID-19 patients. While, laboratory variables in early infection could predict the persistence of viral RNA.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , COVID-19/prevention & control , Cohort Studies , Retrospective Studies , RNA, Viral , Vaccination , Antibodies, ViralABSTRACT
Variants of severe acute respiratory syndrome coronavirus 2 frequently arise within infected individuals. Here, we explored the level and pattern of intra-host viral diversity in association with disease severity. Then, we analyzed information underlying these nucleotide changes to infer the impetus including mutational signatures and immune selection from neutralizing antibody or T-cell recognition. From 23 January to 31 March 2020, a set of cross-sectional samples were collected from individuals with homogeneous founder virus regardless of disease severity. Intra-host single-nucleotide variants (iSNVs) were enumerated using deep sequencing. Human leukocyte antigen (HLA) alleles were genotyped by Sanger sequencing. Medical records were collected and reviewed by attending physicians. A total of 836 iSNVs (3-106 per sample) were identified and distributed in a highly individualized pattern. The number of iSNVs paced with infection duration peaked within days and declined thereafter. These iSNVs did not stochastically arise due to a strong bias toward C > U/G > A and U > C/A > G substitutions in reciprocal proportion with escalating disease severity. Eight nonsynonymous iSNVs in the receptor-binding domain could escape from neutralization, and eighteen iSNVs were significantly associated with specific HLA alleles. The level and pattern of iSNVs reflect the in vivo viral-host interaction and the disease pathogenesis.
ABSTRACT
The origins of preexisting SARS-CoV-2 cross-reactive antibodies and their potential impacts on vaccine efficacy have not been fully clarified. In this study, we demonstrated that S2 was the prevailing target of the preexisting S protein cross-reactive antibodies in both healthy human and SPF mice. A dominant antibody epitope was identified on the connector domain of S2 (1147-SFKEELDKYFKNHT-1160, P144), which could be recognized by preexisting antibodies in both human and mouse. Through metagenomic sequencing and fecal bacteria transplant, we demonstrated that the generation of S2 cross-reactive antibodies was associated with commensal gut bacteria. Furthermore, six P144 reactive monoclonal antibodies were isolated from naïve SPF mice and were proven to cross-react with commensal gut bacteria collected from both human and mouse. A variety of cross-reactive microbial proteins were identified using LC-MS, of which E. coli derived HSP60 and HSP70 proteins were confirmed to be able to bind to one of the isolated monoclonal antibodies. Mice with high levels of preexisting S2 cross-reactive antibodies mounted higher S protein specific binding antibodies, especially against S2, after being immunized with a SARS-CoV-2 S DNA vaccine. Similarly, we found that levels of preexisting S2 and P144-specific antibodies correlated positively with RBD binding antibody titers after two doses of inactivated SARS-CoV-2 vaccination in human. Collectively, our study revealed an alternative origin of preexisting S2-targeted antibodies and disclosed a previously neglected aspect of the impact of gut microbiota on host anti-SARS-CoV-2 immunity.
Subject(s)
COVID-19 , Gastrointestinal Microbiome , Viral Vaccines , Animals , Antibodies, Monoclonal , Antibodies, Viral , COVID-19/prevention & control , COVID-19 Vaccines , Escherichia coli , Humans , Mice , SARS-CoV-2ABSTRACT
Interleukin-12 receptor ß1 (IL12RB1)-deficient individuals show increased susceptibilities to local or disseminated BCG infection and environmental mycobacteria infection. However, the low clinical penetrance of IL12RB1 deficiency and low recurrence rate of mycobacteria infection suggest that protective immunity still exists in this population. In this study, we investigated the mechanism of tuberculosis suppression using the IL12RB1-deficient mouse model. Our results manifested that Il12rb1-/- mice had significantly increased CFU counts in spleens and lungs, especially when BCG (Danish strain) was inoculated subcutaneously. The innate TNF-a and IFN-γ responses decreased, while the IL-17 responses increased significantly in the lungs of Il12rb1-/- mice. We also found that PPD-specific IFN-γ release was impaired in Il12rb1-/- mice, but the specific TNF-a release was not compromised, and the antibody responses were significantly enhanced. Moreover, correlation analyses revealed that both the innate and PPD-specific IFN-γ responses positively correlated with CFU counts, whereas the innate IL-12a levels negatively correlated with CFU counts in Il12rb1-/- mice lungs. Collectively, these findings proved that the adaptive immunities against mycobacteria are not completely nullified in Il12rb1-/- mice. Additionally, our results imply that IFN-γ responses alone might not be able to contain BCGitis in the setting of IL12RB1 deficiency.
ABSTRACT
Cryptococcosis is an opportunistic and potentially lethal infection caused by Cryptococcus neoformans and Cryptococcus gattii complex, which affects both immunocompromised and immunocompetent people, and it has become a major public health concern worldwide. In this study, we characterized the molecular epidemiology and antifungal susceptibility of 133 C. neoformans isolates from East China Invasive Fungal Infection Group (ECIFIG), 2017-2020. Isolates were identified to species level by matrix-assisted laser desorption ionization-time of flight mass spectrometry and confirmed by IGS1 sequencing. Whole-genome sequencing (WGS) was conducted on three multidrug-resistant isolates. Among the 133 strains, 61 (45.86%) were isolated from HIV-positive patients and 72 (54.16%) were isolated from HIV-negative patients. In total, C. neoformans var. grubii accounted for 97.74% (130/133), while C. neoformans var. neoformans was rare (2.06%, 3/133). The strains were further classified into nine sequence types (STs) dominated by ST5 (90.23%, 120/133) with low genetic diversity. No association was observed between STs and HIV status. All strains were wild type to voriconazole, while high antifungal minimal inhibitory concentrations (MICs) above the epidemiological cutoff values (ECVs) were observed in C. neoformans strains, and more than half of isolates were non-wild-type to amphotericin B (89.15%, 109/133). Eight isolates were resistant to fluconazole, and eight isolates were non-wild type to 5-fluorocytosine. Furthermore, WGS has verified the novel mutations of FUR1 in 5-fluorocytosine-resistant strains. In one isolate, aneuploidy of chromosome 1 with G484S mutation of ERG11 was observed, inducing high-level resistance (MIC: 32 µg/ml) to fluconazole. In general, our data showed that there was no significant difference between HIV-positive and HIV-negative patients on STs, and we elucidate the resistant mechanisms of C. neoformans from different perspectives. It is important for clinical therapy and drug usage in the future.
ABSTRACT
OBJECTIVE: Cell metabolism plays a vital role in the proliferation, metastasis and sensitivity to chemotherapy drugs of colorectal cancer. The purpose of this multicenter cohort study is to investigate the potential genes indicating clinical outcomes in colorectal cancer patients. METHODS: We analyzed gene expression profiles of colorectal cancer to identify differentially expressed genes then used these differentially expressed genes to construct prognostic signature based on the least absolute shrink-age and selection operator Cox regression model. In addition, the multi-gene signature was validated in independent datasets including our multicenter cohort. Finally, nomograms were set up to evaluate the prognosis of colorectal cancer patients. RESULTS: Seventeen metabolism-related genes were determined in the least absolute shrink-age and selection operator model to construct signature, with area under receiver operating characteristic curve for relapse-free survival, 0.741, 0.755 and 0.732 at 1, 3 and 5 year, respectively. External validation datasets, GSE14333, GSE37892, GSE17538 and the Cancer Genome Atlas cohorts, were analyzed and stratified, indicating that the metabolism-related signature was reliable in discriminating high- and low-risk colorectal cancer patients. Area under receiver operating characteristic curves for relapse-free survival in our multicenter validation cohort were 0.801, 0.819 and 0.857 at 1, 3 and 5 year, respectively. Nomograms incorporating the genetic biomarkers and clinical pathological features were set up, which yielded good discrimination and calibration in the prediction of prognosis for colorectal cancer patients. CONCLUSION: An original metabolism-related signature was developed as a predictive model for the prognosis of colorectal cancer patients. A nomogram based on the signature was advantageous to facilitate personalized counselling and treatment of colorectal cancer patients.
Subject(s)
Colorectal Neoplasms , Gene Expression Regulation, Neoplastic , Humans , Cohort Studies , Prognosis , Nomograms , Colorectal Neoplasms/genetics , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolismABSTRACT
A variety of methods have been explored to increase delivery efficiencies for DNA vaccine. However, the immunogenicity of DNA vaccines has not been satisfactorily improved. Unlike most of the previous attempts, we provided evidence suggesting that changing the injection site successively (successively site-translocated inoculation, SSTI) could significantly enhance the immunogenicity of DNA vaccines in a previous study. To simplify the strategy and to evaluate its impact on candidate SARS-CoV-2 vaccines, we immunized mice with either a SARS-CoV-2 spike-based DNA vaccine or a spike protein subunit vaccine via three different inoculation strategies. Our data demonstrated that S protein specific antibody responses elicited by the DNA vaccine or the protein subunit vaccine showed no significant difference among different inoculation strategies. Of interest, compared with the conventional site fixed inoculation (SFI), both successive site-translocating inoculation (SSTI) and the simplified translocating inoculation (STI) strategy improved specific T cell responses elicited by the DNA vaccine. More specifically, the SSTI strategy significantly improved both the monofunctional (IFN-γ+IL-2-TNF-α-CD8+) and the multifunctional (IFN-γ+IL-2-TNF-α+CD8+, IFN-γ+IL-2-TNF-α+CD4+, IFN-γ+IL-2+TNF-α+CD4+) T cell responses, while the simplified translocating inoculation (STI) strategy significantly improved the multifunctional CD8+ (IFN-γ+IL-2-TNF-α+CD8+, IFN-γ+IL-2+TNF-α+CD8+) and CD4+ (IFN-γ+IL-2-TNF-α+CD4+, IFN-γ+IL-2+TNF-α+CD4+) T cell responses. The current study confirmed that changing the site of intra muscular injection can significantly improve the immunogenicity of DNA vaccines.
Subject(s)
COVID-19 , Sexually Transmitted Diseases , Vaccines, DNA , Animals , CD4-Positive T-Lymphocytes , CD8-Positive T-Lymphocytes , COVID-19/prevention & control , COVID-19 Vaccines , Humans , Interleukin-2 , Mice , Protein Subunits , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Tumor Necrosis Factor-alphaABSTRACT
Tryptophan (Trp) as an essential amino acid plays critical roles in regulating multiple cell activities, and the changes of its circulating level usually indicate disease status such as the severity of acute inflammation sepsis. However, the current technology for Trp detection mostly relies on chromatography that cannot meet the rapid and simple detection requirement in monitoring sepsis. Herein, a label-free fluorescent nanosensor was constructed to detect Trp and its carrier protein - human serum albumin (HSA) in plasma. The nanosensor consists of a cytosine (C) - rich signal probe of DNA-templated silver nanoclusters (AgNCs/DNA) and a Trp (Trp) aptamer - based capture probe with a guanine (G) - rich overhang. Trp was found to trigger G-rich sequence-mediated fluorescence enhancement effect for AgNCs/DNA under UV irradiation which offers energy to induce the redox process between limited Trp and silver ions bound on the DNA probes. Based on this photochemical property, the nanosensor exhibited a linear response in the range of 0.05-60 µM with the limit of detection of 0.43 µM for Trp, superior to the current fluorescence-based detection method, and it gave specific response to indole group. When applied in plasma detection, the nanosensor resisted physiological level of NaCl in plasma, but was quenched by trace volume of HSA, which facilitates the combined HSA detection using only 1 µL plasma sample. The simple procedure of "mix, exposure and detection" together with its ultralow sampling volume, time-saving, cost-effective, sensitive and selective properties endow the nanosensor great potentials for future Trp detection-based clinical use.
Subject(s)
Metal Nanoparticles , Sepsis , Albumins , DNA/chemistry , Fluorescent Dyes/chemistry , Humans , Metal Nanoparticles/chemistry , Sepsis/diagnosis , Silver/chemistry , TryptophanABSTRACT
Hepatitis B virus exposure in children usually develops into chronic hepatitis B (CHB). Although hepatitis B surface antigen (HBsAg)-specific CD8+ T cells contribute to resolve HBV infection, they are preferentially undetected in CHB patients. Moreover, the mechanism for this rarely detected HBsAg-specific CD8+ T cells remains unexplored. We herein found that the frequency of HBsAg-specific CD8+ T cells was inversely correlated with expansion of monocytic myeloid-derived suppressor cells (mMDSCs) in young rather than in adult CHB patients, and CCR9 was upregulated by HBsAg on mMDSCs via activation of ERK1/2 and IL-6. Sequentially, the interaction between CCL25 and CCR9 mediated thymic homing of mMDSCs, which caused the cross-presentation, transferring of peripheral HBsAg into the thymic medulla, and then promoted death of HBsAg-specific CD8+ thymocytes. In mice, adoptive transfer of mMDSCs selectively obliterated HBsAg-specific CD8+ T cells and facilitated persistence of HBV in a CCR9-dependent manner. Taken together, our results uncovered a novel mechanism for establishing specific CD8+ tolerance to HBsAg in chronic HBV infection.
Subject(s)
Hepatitis B, Chronic , Myeloid-Derived Suppressor Cells , Animals , CD8-Positive T-Lymphocytes , Hepatitis B Surface Antigens , Hepatitis B virus , Humans , MiceABSTRACT
The detection of samples at ultralow concentrations (one to ten copies in 100 µl) in biofluids is hampered by the orders-of-magnitude higher amounts of 'background' biomolecules. Here we report a molecular system, immobilized on a liquid-gated graphene field-effect transistor and consisting of an aptamer probe bound to a flexible single-stranded DNA cantilever linked to a self-assembled stiff tetrahedral double-stranded DNA structure, for the rapid and ultrasensitive electromechanical detection (down to one to two copies in 100 µl) of unamplified nucleic acids in biofluids, and also of ions, small molecules and proteins, as we show for Hg2+, adenosine 5'-triphosphate and thrombin. We implemented an electromechanical biosensor for the detection of SARS-CoV-2 into an integrated and portable prototype device, and show that it detected SARS-CoV-2 RNA in less than four minutes in all nasopharyngeal samples from 33 patients with COVID-19 (with cycle threshold values of 24.9-41.3) and in none of the 54 COVID-19-negative controls, without the need for RNA extraction or nucleic acid amplification.
Subject(s)
COVID-19 , Graphite , COVID-19/diagnosis , Humans , Ions , RNA, Viral/genetics , SARS-CoV-2/geneticsSubject(s)
COVID-19 , SARS-CoV-2 , Genome, Viral , Humans , Mutation , Nucleotides , Pandemics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/geneticsABSTRACT
COVID-19 continues to circulate globally in 2021, while under the precise policy implementation of China's public health system, the epidemic was quickly controlled, and society and the economy have recovered. During the pandemic response, nucleic acid detection of SARS-CoV-2 has played an indispensable role in the first line of defence. In the cases of emergency operations or patients presenting at fever clinics, nucleic acid detection is required to be performed and reported quickly. Therefore, nucleic acid point-of-care testing (POCT) technology for SARS-CoV-2 identification has emerged, and has been widely carried out at all levels of medical institutions. SARS-CoV-2 POCT has served as a complementary test to conventional polymerase chain reaction (PCR) batch tests, thus forming an experimental diagnosis platform that not only guarantees medical safety but also improves quality services. However, in view of the complexity of molecular diagnosis and the biosafety requirements involved, pathogen nucleic acid POCT is different from traditional blood-based physical and chemical index detection. No guidelines currently exist for POCT quality management, and there have been inconsistencies documented in practical operation. Therefore, Shanghai Society of Molecular Diagnostics, Shanghai Society of Laboratory Medicine, Clinical Microbiology Division of Shanghai Society of Microbiology and Shanghai Center for Clinical Laboratory have cooperated with experts in laboratory medicine to generate the present expert consensus. Based on the current spectrum of major infectious diseases in China, the whole-process operation management of pathogen POCT, including its application scenarios, biosafety management, personnel qualification, performance verification, quality control, and result reporting, are described here. This expert consensus will aid in promoting the rational application and robust development of this technology in public health defence and hospital infection management.
Subject(s)
COVID-19 , Nucleic Acids , China , Consensus , Humans , Point-of-Care Testing , SARS-CoV-2ABSTRACT
Direct SARS-CoV-2 nucleic acid testing with fast speed and high frequency is crucial for controlling the COVID-19 pandemic. Here, direct testing of SARS-CoV-2 nucleic acid is realized by field-effect transistors (FETs) with an electro-enrichable liquid gate (LG) anchored by tetrahedral DNA nanostructures (TDNs). The applied gate bias electrostatically preconcentrates nucleic acids, while the liquid gate with TDNs provides efficient analyte recognition and signal transduction. The average diagnosis time is â¼80 s, and the limit of detection approaches 1-2 copies in 100 µL of clinical samples without nucleic acid extraction and amplification. As such, TDN-LG FETs solve the dilemma of COVID-19 testing on mass scale that diagnosis accuracy and speed undergo trade-off. In addition, TDN-LG FETs achieve unamplified 10-in-1 pooled nucleic acid testing for the first time, and the results are consistent with PCR. Thus, this technology promises on-site and wide population COVID-19 screening and ensures safe world-reopening.