Tuberculosis (TB) is a serious, potentially fatal disease that has been a global public health issue for centuries. The growth of the causal bacterium Mycobacterium tuberculosis (M. tuberculosis) is ...slow (at least 6–8 weeks) when compared to other infectious bacteria, thus posing a challenge for early diagnostics. Furthermore, TB therapy has been reported to be effective when antibiotics are administered during the early stage. Therefore, accurate and rapid diagnosis of M. tuberculosis plays an important role in TB quarantine and treatment. We therefore devised an integrated microfluidic system to automatically detect live M. tuberculosis and distinguish dead bacteria. A heparin-binding hemagglutinin antibody was used to capture bacteria within 10 min. The photo-reactive dye propidium monoazide was then used to bind double-stranded DNA from dead bacteria with high affinity (20 min) such that genes of dead bacteria were not amplified by the subsequent PCR. Additionally, after 10-min of genomic DNA isolation, the final PCR step (50 min) was carried out to detect live M. tuberculosis. It is the first time that the entire bacterial detection process (including bacterial capture, propidium monoazide treatment, lysis and PCR quantification) for live M. tuberculosis could be automated within 90 min on a single chip with a limit of detection as low as 100 colony-forming units. This integrated microfluidic system is promising for point-of-care diagnostics given its automation, high speed, low cost, low sample/reagent consumption, and reduced risk of infection for front-line medical staff.
•An integrated microfluidic system to automatically detect live M. tuberculosis and distinguish dead bacteria was developed.•The entire bacterial detection process (including bacterial capture, propidium monoazide treatment, lysis and PCR quantification) for live M. tuberculosis could be automated within 90 min on a single chip.•A limit of detection as low as 100 colony-forming units was found.
The development of efficient vaccines against COVID-19 is an emergent need for global public health. The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major ...target for the COVID-19 vaccine. To quickly respond to the outbreak of the SARS-CoV-2 pandemic, a nucleic acid-based vaccine is a novel option, beyond the traditional inactivated virus vaccine or recombinant protein vaccine. Here, we report a DNA vaccine containing the spike gene for delivery via electroporation. The spike genes of SARS-CoV and SARS-CoV-2 were codon optimized for mammalian cell expression and then cloned into mammalian cell expression vectors, called pSARS-S and pSARS2-S, respectively. Spike protein expression was confirmed by immunoblotting after transient expression in HEK293T cells. After immunization, sera were collected for antigen-specific antibody and neutralizing antibody titer analyses. We found that both pSARS-S and pSARS2-S immunization induced similar levels of antibodies against S2 of SARS-CoV-2. In contrast, only pSARS2-S immunization induced antibodies against the receptor-binding domain of SARS-CoV-2. We further found that pSARS2-S immunization, but not pSARS-S immunization, could induce very high titers of neutralizing antibodies against SARS-CoV-2. We further analyzed SARS-CoV-2 S protein-specific T cell responses and found that the immune responses were biased toward Th1. Importantly, pSARS2-S immunization in hamsters could induce protective immunity against SARS-CoV-2 challenge in vivo. These data suggest that DNA vaccination could be a promising approach for protecting against COVID-19.
Pulmonary granuloma diseases caused by
(
) have increased in past decades, and drug-resistance in this pathogen is a growing public health concern. Therefore, an animal model of chronic granuloma ...disease is urgently needed.
In this study,
embedded within agar beads (agar-AB) was used to develop such a model in C57BL/6JNarl mice.
Chronic infection was sustained for at least 3 months after agar-AB infection, visible granulomas spread in the lungs, and giant cells and foamy cells appeared in the granulomas. More importantly, pulmonary fibrosis progressed for 3 months, and collagen fibers were detected by Masson trichrome staining. Further, inducible nitric oxide synthase (iNOS) was highly expressed within the alveolar space, and the fibrosis-mediator transforming growth factor beta (TGF-β) began to be expressed at 1 month. Hypoxia-inducible factor (HIF-1α) expression also increased, which aided in normalizing oxygen partial pressure.
Although the transient fibrosis persisted for only 3 months, and the pulmonary structure resolved when the pathogen was cleard, this pulmonary fibrosis model for
infection will provide a novel test platform for development of new drugs, regimens, and therapies.
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•A multi-labeled electrochemical sensor containing two universal probes is demonstrated for cost-efficient analysis of single nucleotide substitutions (SNS).•The sensor is capable of ...detecting SNS in arbitrary and folded nucleic acids.•The ability differentiating drug-resistant from drug-susceptible M.tb is demonstrated.
Analysis of single nucleotide substitutions (SNS) in nucleic acids is the basis for the diagnosis of drug-resistant pathogens as well as human genetic disorders among a broad range of other applications. However, SNS are often inaccessible for the analysis by hybridization probe due to the location in double stranded regions of folded single stranded DNA or RNA analytes. Here we introduce an electrochemical sensor that is able to analyze SNS in folded nucleic acids. The electrochemical sensor takes advantage of a universal electrode-bound hairpin (UTH). It relies on the recognition of targeted nucleic acids by analyte-specific adaptor strands R and L that also hybridize with UTH. Strand L can bind several methylene blue (MeB)-probes thus placing MeB groups close to the electrode surface, which enables cathodic charge transfer. We demonstrated that the same UTH-functionalized electrode and MeB-probe can be used to analyze DNA analytes with different sequences. The sensor is highly selective toward SNS even at room temperature and can be regenerated for next round by rinse with water. The ability of the sensor to analyze SNS within secondary structure folded DNA was demonstrated. The sensing system is capable of detecting SNS in bacterial DNA, useful to differentiate drug-resistant from drug-susceptible mycobacterium tuberculosis. The proposed platform uniquely combines high SNS selectivity with ability to analyze potentially any DNA or RNA sequence including those folded in stable structures, therefore, creates a basis for a cost efficient electrochemical sensing of nucleic acids applicable both in medical diagnostics and environmental monitoring of microorganisms.
A series of recombinant human type 5 adenoviruses that express the full-length or membrane-truncated spike protein (S) of SARS-CoV-2 (AdCoV2-S or AdCoV2-SdTM, respectively) was tested the efficacy ...against SARS-CoV-2 via intranasal (i.n.) or subcutaneous (s.c.) immunization in a rodent model. Mucosal delivery of adenovirus (Ad) vaccines could induce anti-SARS-CoV-2 IgG and IgA in the serum and in the mucosal, respectively as indicated by vaginal wash (vw) and bronchoalveolar lavage fluid (BALF). Serum anti-SARS-CoV-2 IgG but not IgA in the vw and BALF was induced by AdCoV2-S s.c.. Administration of AdCoV2-S i.n. was able to induce higher anti-SARS-CoV-2 binding and neutralizing antibody levels than s.c. injection. AdCoV2-SdTM i.n. induced a lower antibody responses than AdCoV2-S i.n.. Induced anti-S antibody responses by AdCoV2-S via i.n. or s.c. were not influenced by the pre-existing serum anti-Ad antibody. Novelty, S-specific IgG1 which represented Th2-mediated humoral response was dominantly induced in Ad i.n.-immunized serum in contrast to more IgG2a which represented Th1-mediated cellular response found in Ad s.c.-immunized serum. The activation of S-specific IFN-ɣ and IL-4 in splenic Th1 and Th2 cells, respectively, was observed in the AdCoV2-S i.n. and s.c. groups, indicating the Th1 and Th2 immunity were activated. AdCoV2-S and AdCoV2-SdTM significantly prevented body weight loss and reduced pulmonary viral loads in hamsters. A reduction in inflammation in the lungs was observed in AdCoV-S via i.n. or s.c.-immunized hamsters following a SARS-CoV-2 challenge. It correlated to Th1 cytokine but no inflammatory cytokines secretions found in AdCoV-S i.n. -immunized BALF. These results indicate that intranasal delivery of AdCoV2-S vaccines is safe and potent at preventing SARS-CoV-2 infections.
Pulmonary tuberculosis (TB) is a difficult-to-eliminate disease. Although the Bacille Calmette-Guérin (BCG) vaccine against
(MTB) has been available for decades, its efficacy is variable and has ...lessened over time. Furthermore, the BCG vaccine no longer protects against newly emerged Beijing strains which are responsible for many current infections in adults. Development of a novel vaccine is urgently needed. In this study, we first tested the efficacy of our recombinant BCG vaccines rBCG1 and rBCG2, compared to parental BCG, against MTB strain H37Ra in mice. Both the bacterial load and the level of lymphocyte infiltration decreased dramatically in the three groups treated with vaccine, especially rBCG1 and rBCG2. Furthermore, the Th1 and Th17 responses increased and macrophage numbers rose in the vaccination groups. Th1-mediated production of cytokines TNF-α, IFN-γ, and MCP-1 as well as M1-polarized cells all increased in lung tissue of the rBCG1 and rBCG2 groups. Clodronate-induced depletion of macrophages reduced the level of protection. Based on these results, we conclude that rBCG vaccines induce a significant increase in the number of M1 macrophages, which augments their potential as TB vaccine candidates.
Tuberculosis (TB) is a leading cause of death from a single infectious agent,
(
). Although progress has been made in TB control, still about 10 million people worldwide develop TB annually and 1.5 ...million die of the disease. The rapid emergence of aggressive, drug-resistant strains and latent infections have caused TB to remain a global health challenge. TB treatments are lengthy and their side effects lead to poor patient compliance, which in turn has contributed to the drug resistance and exacerbated the TB epidemic. The relatively low output of newly approved antibiotics has spurred research interest toward alternative antibacterial molecules such as silver nanoparticles (AgNPs). In the present study, we use the natural biopolymer alginate to serve as a stabilizer and/or reductant to green synthesize AgNPs, which improves their biocompatibility and avoids the use of toxic chemicals. The average size of the alginate-capped AgNPs (ALG-AgNPs) was characterized as nanoscale, and the particles were round in shape. Drug susceptibility tests showed that these ALG-AgNPs are effective against both drug-resistant
strains and dormant
. A bacterial cell-wall permeability assay showed that the anti-mycobacterial action of ALG-AgNPs is mediated through an increase in cell-wall permeability. Notably, the anti-mycobacterial potential of ALG-AgNPs was effective in both zebrafish and mouse TB animal models
. These results suggest that ALG-AgNPs could provide a new therapeutic option to overcome the difficulties of current TB treatments.
It is unclear to what extent the host-responses elicited by Beijing versus non-Beijing strains of Mycobacterium tuberculosis (MTB) contribute to the predominance of modern Beijing strains in Taiwan ...and some other Asian countries. The purpose of this study was to compare the expression profiles of virulence-related genes in human monocyte-derived macrophages infected in vitro with Beijing (ancient and modern strains) and non-Beijing strains (EAI strains) of MTB that are epidemic in Taiwan. We found that modern Beijing strains induced lower levels of pro-inflammatory cytokines, whereas EAI strains induced higher levels. Notably, the most prevalent modern Beijing sub-lineage, possessing intact RD150 and RD142 chromosomal regions, induced very low levels of pro-inflammatory cytokines, especially interleukin-1β. Moreover, in an intracellular growth assay, the survival of the same modern Beijing strain in human monocyte-derived macrophages was significantly higher than that of an ancient Beijing strain and an EAI strain. Taken together, these results may explain why modern Beijing strains of MTB predominate in Taiwan.
Background/Purpose A globally increasing trend of type 2 diabetes mellitus (DM), the rising prevalence of tuberculosis (TB) in many countries, and the emergence of multidrug-resistant TB (MDR-TB) in ...recent years pose a serious challenge for TB control. Methods We investigated pulmonary tuberculosis patients with and without type 2 DM (DMTB and TB, respectively) treated at the Chest Hospital, Taiwan, between November 2004 and October 2005. Results One hundred and ninety-two new patients (60 DMTB, 132 TB) were regularly treated for a full course (≥ 6 months) and prospectively followed for more than 1 year. The DMTB patients had more severe infections (far-advanced: 45.0% vs. 22.7%, p < 0.01), higher mycobacterial loads (sputum smear: 2.9 ± 1.3+ vs. 1.9 ± 1.7+ , p < 0.01), higher treatment failure rates (17% vs. 2%, p < 0.01), and longer delayed clearance of mycobacteria than did the TB patients (2.5 ± 3.0 months vs. 1.6 ± 1.4 months, p < 0.01). After one year, three DMTB patients and one TB patient had MDR-TB (5.0% vs. 0.8%, p = 0.056). Bacterial genotyping revealed that the proportion of mycobacterial strains was not significantly different in DMTB and TB patients (Beijing strain: 46.7% vs. 40.6%, Non-Beijing strain: 53.3% vs. 59.4%, p = 0.632). Conclusion DMTB patients have more severe TB infections, which require longer treatment and are more likely to develop MDR-TB than are patients with TB alone.
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