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Development and evaluation of a triplex droplet digital PCR method for differentiation of , and BCG.Frontiers in Microbiology 2024Tuberculosis, caused by complex (MTBC), remains a global health concern in both human and animals. However, the absence of rapid, accurate, and highly sensitive...
INTRODUCTION
Tuberculosis, caused by complex (MTBC), remains a global health concern in both human and animals. However, the absence of rapid, accurate, and highly sensitive detection methods to differentiate the major pathogens of MTBC, including , , and BCG, poses a potential challenge.
METHODS
In this study, we have established a triplex droplet digital polymerase chain reaction (ddPCR) method employing three types of probe fluorophores, with targets (targeting CFP-10-ESAT-6 gene of RD1 and Rv0222 genes of RD4), (targeting CFP-10-ESATs-6 gene of RD1), and BCG (targeting Rv3871 and Rv3879c genes of ΔRD1), respectively.
RESULTS
Based on optimization of annealing temperature, sensitivity and repeatability, this method demonstrates a lower limit of detection (LOD) as 3.08 copies/reaction for , 4.47 copies/reaction for and 3.59 copies/reaction for BCG, without cross-reaction to , , , , , , , , and , and showed repeatability with coefficients of variation (CV) lower than 10%. The method exhibits strong milk sample tolerance, the LOD of detecting in spike milk was 5 × 10 CFU/mL, which sensitivity is ten times higher than the triplex qPCR. 60 clinical DNA samples, including 20 milk, 20 tissue and 20 swab samples, were kept in China Animal Health and Epidemiology Center were tested by the triplex ddPCR and triplex qPCR. The triplex ddPCR presented a higher sensitivity (11.67%, 7/60) than that of the triplex qPCR method (8.33%, 5/60). The positive rates of , , and BCG were 1.67, 10, and 0% by triplex ddPCR, and 1.67, 6.67, and 0% by triplex qPCR, with coincidence rates of 100, 96.7, and 100%, respectively.
DISCUSSION
Our data demonstrate that the established triplex ddPCR method is a sensitive, specific and rapid method for differentiation and identification of , , and BCG.
PubMed: 38946908
DOI: 10.3389/fmicb.2024.1397792 -
Tropical Diseases, Travel Medicine and... Jul 2024Tuberculosis (TB) is a major public health concern in the developing countries. Moreover, the emergence of multidrug-resistant tuberculosis is challenging. However,...
BACKGROUND
Tuberculosis (TB) is a major public health concern in the developing countries. Moreover, the emergence of multidrug-resistant tuberculosis is challenging. However, there are no organized data on the trends of pulmonary tuberculosis and rifampicin-resistant Mycobacterium tuberculosis in the study area.
METHODS
A retrospective cross-sectional study was conducted to fill the information gap in Central Tigray at St. Mary General Hospital between 2018 and 2023. Data were collected from the GeneXpert™ tuberculosis registration logbooks using standard checklists and analyzed using Statistical Package for Social Science version 22. After performing logistic regression, a p-value < 0.05 with a corresponding 95% confidence interval was considered statistically significant. Moreover, chi square test for trend was performed to assess the percentage of annual detection of pulmonary tuberculosis and rifampicin-resistant Mycobacterium tuberculosis during the study years.
RESULT
Presumptive pulmonary tuberculosis patients with complete data (n = 3696) were included in the study. The overall prevalence of pulmonary tuberculosis was 11.7%, of which 8.1% were resistant to rifampicin. The study revealed that the incidence of pulmonary tuberculosis has been increasing, mainly in the recent four years. Likewise, an increase in rifampicin-resistant Mycobacterium tuberculosis was observed with considerable fluctuations. Age, human immunodeficiency virus infection, and presumptive rifampicin-resistant Mycobacterium tuberculosis infection were significantly associated with the presence of pulmonary tuberculosis. Moreover, pulmonary tuberculosis was more prevalent among participants in the productive-age group.
CONCLUSION
Although there have been fluctuations, an increasing of pulmonary tuberculosis and rifampicin-resistant Mycobacterium tuberculosis has been observed in recent years. Hence, prevention and treatment strategies for tuberculosis should be strengthened to alleviate the burden of pulmonary tuberculosis and rifampicin-resistant Mycobacterium tuberculosis in the study area.
PubMed: 38946006
DOI: 10.1186/s40794-024-00224-1 -
American Journal of Infection Control Jun 2024The mounted UV-C lamp requires no separate space or labor for transport, but data on its effectiveness for surface disinfection are scarce. We aimed to evaluate the...
BACKGROUND
The mounted UV-C lamp requires no separate space or labor for transport, but data on its effectiveness for surface disinfection are scarce. We aimed to evaluate the performance of ceiling-mounted UV-C lamps.
METHODS
This study was conducted in an empty room with UV-C lamps in the biocontainment unit of a tertiary care hospital in South Korea. UV-C lamps were installed on the ceiling of the patient room, anteroom, and toilet. Each pathogen (Staphylococcus aureus, Escherichia coli, Candida krusei, Bacillus cereus, and Mycobacterium peregrinum) was inoculated on blood agar plates and placed in 20 selected places from the UV-C lamp, and irradiation was applied for 15 min. As a control group, the bacterial solution was diluted 10,000 times and UV was not applied.
RESULTS
A mean ± SD of 5.95 ± 0.91 log reduction was observed with UV irradiation compared with the control. The log reduction was greatest for S. aureus [median, 7.05 (IQR, 6.49-7.26)] and least for M. peregrinum [median, 4.88 (IQR, 4.58-5.24)]. The degree of log reduction was inversely proportional to the square of the distance from the UV-C lamp (R = -0.12, p < 0.001).
CONCLUSIONS
In this study, ceiling-mounted UV-C demonstrated effective disinfection of at least 4-log reduction of the test organisms within a 4-m distance. Mounted UV-C lighting is a considerable option for improving surface disinfection.
PubMed: 38945300
DOI: 10.1016/j.ajic.2024.06.020 -
Journal of Hazardous Materials Jun 2024Natural attenuation (NA) is of great significance for the remediation of contaminated groundwater, and how to identify NA patterns of toluene in aquifers more quickly...
Natural attenuation (NA) is of great significance for the remediation of contaminated groundwater, and how to identify NA patterns of toluene in aquifers more quickly and effectively poses an urgent challenge. In this study, the NA of toluene in two typical soils was conducted by means of soil column experiment. Based on column experiments, dissolved organic matter (DOM) was rapidly identified using fluorescence spectroscopy, and the relationship between DOM and the NA of toluene was established through structural equation modeling analysis. The adsorption rates of toluene in clay and sandy soil were 39 % and 26 %, respectively. The adsorption capacity and total NA capacity of silty clay were large. The occurrence of fluorescence peaks of protein-like components and specific products indicated the occurrence of biodegradation. Arenimonas, Acidovorax and Brevundimonas were the main degrading bacteria identified in Column A, while Pseudomonas, Azotobacter and Mycobacterium were the main ones identified in Column B. The pH, ORP, and Fe(II) were the most important factors affecting the composition of microbial communities, which in turn affected the NA of toluene. These results provide a new way to quickly identify NA of toluene.
PubMed: 38944985
DOI: 10.1016/j.jhazmat.2024.134952 -
International Journal of Pediatric... Jun 2024Cervicofacial lymphadenitis caused by non-tubercular mycobacterial (NTM) infections has the highest infection rate in children. Our objective was to assess patient...
OBJECTIVE
Cervicofacial lymphadenitis caused by non-tubercular mycobacterial (NTM) infections has the highest infection rate in children. Our objective was to assess patient demographics, treatment methods, and the impact of weather and geography on the incidence of disease in patients with NTM cervicofacial lymphadenitis.
METHODS
The Pediatric Health Information System (PHIS) database was queried for data on all patients diagnosed with concurrent cervicofacial lymphadenopathy and NTM infection from 2004 to 2022. We assessed the association between weather patterns and NTM cervicofacial lymphadenitis by collecting monthly weather data from the NOAA National Center for Environmental Information. Incidence rates were calculated by dividing the number of cases by the total hospital discharges during the study period.
RESULTS
Among 47 PHIS hospitals, there were 992 diagnoses of NTM cervicofacial lymphadenitis. The average age at diagnosis was 2 [IQR, 2-4], with 59 % female. Drainage of skin abscesses or lesions was performed for 93 (9.4 %) patients, while 15 (1.5 %) had an excisional procedure of the CPT codes assessed. The most common antibiotics utilized were cephalosporins (28 %), macrolides (27 %), and rifampin (12 %). The most common treatment method was surgery with antibiotics (37 %) followed by no treatment at all (35 %), surgery alone (17 %), and antibiotics alone (10 %). Of the 28 states included in the analysis, Washington (IR: 3.5) and Nebraska (IR: 3.3) had the highest incidence rates (IR) of NTM cervical lymphadenitis. The cases were relatively equally distributed across the different weather seasons within each U.S. geographic region. However, the overall average wind speed was weakly associated with increasing the risk of diagnosis when utilizing a mixed effect zero-inflated negative binomial model (Incidence Ratio: 1.07, 95 % CI: (1.01-1.14), p = 0.035).
CONCLUSIONS
Our results indicate that the most common treatment method utilized in patients within our cohort with NTM cervicofacial lymphadenitis was the concurrent use of surgery and antibiotics. Our results also indicate there may be variation in the incidence rate among different states, but additional studies are needed as our cohort only included approximately 50 % of states within the U.S.
PubMed: 38944979
DOI: 10.1016/j.ijporl.2024.112019 -
International Immunopharmacology Jun 2024Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), still ranks among the leading causes of annual human death by infectious disease. Mtb has developed several...
Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), still ranks among the leading causes of annual human death by infectious disease. Mtb has developed several strategies to survive for years at a time within the host despite the presence of a robust immune response, including manipulating the progression of the inflammatory response and forming granulomatous lesions. Here we demonstrate that IQGAP1, a highly conserved scaffolding protein, compartmentalizes and coordinates multiple signaling pathways in macrophages infected with Mycobacterium marinum (Mm or M.marinum), the closest relative of Mtb. Upregulated IQGAP1 ultimately suppresses TNF-α production by repressing the MKK3 signal and reducing NF-κBp65 translocation, deactivating the p38MAPK pathway. Accordingly, IQGAP1 silencing and overexpression significantly alter p38MAPK activity by modulating the production of phosphorylated MKK3 during mycobacterial infection. Pharmacological inhibition of IQGAP1-associated microtubule assembly not only alleviates tissue damage caused by M.marinum infection but also significantly decreases the production of VEGF-a critical player for granuloma-associated angiogenesis during pathogenic mycobacterial infection. Similarly, IQGAP1 silencing in Mm-infected macrophages diminishes VEGF production, while IQGAP1 overexpression upregulates VEGF. Our data indicate that mycobacteria induce IQGAP1 to hijack NF-κBp65 activation, preventing the expression of proinflammatory cytokines as well as promoting VEGF production during infection and granuloma formation. Thus, therapies targeting host IQGAP1 may be a promising strategy for treating tuberculosis, particularly in drug-resistant diseases.
PubMed: 38944950
DOI: 10.1016/j.intimp.2024.112549 -
Journal of Ethnopharmacology Jun 2024African wormwood (Artemisia afra Jacq. ex Willd.) has been used traditionally in southern Africa to treat illnesses causing fever and was recently shown to possess...
ETHNOPHARMACOLOGICAL RELEVANCE
African wormwood (Artemisia afra Jacq. ex Willd.) has been used traditionally in southern Africa to treat illnesses causing fever and was recently shown to possess anti-tuberculosis activity. As tuberculosis is an endemic cause of fever in southern Africa, this suggests that the anti-tubercular activity of A. afra may have contributed to its traditional medicinal use.
AIM OF THE STUDY
Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is a deadly and debilitating disease globally affecting millions annually. Emerging drug-resistant Mtb strains endanger the efficacy of the current therapies employed to treat tuberculosis; therefore, there is an urgent need to develop novel drugs to combat this disease. Given the reported activity of A. afra against Mtb, we sought to determine the mechanisms by which A. afra inhibits and kills this bacterium.
MATERIALS AND METHODS
We used transcriptomics to investigate the impact of Artemisia spp. extracts on Mtb physiology. We then used chromatographic fractionation and biochemometric analyses to identify a bioactive fractions of A. afra extracts and identify an active compound.
RESULTS
Transcriptomic analysis revealed that A. afra exerts different effects on Mtb compared to A. annua or artemisinin, suggesting that A. afra possesses other phytochemicals with unique modes of action. A biochemometric study of A. afra resulted in the isolation of an O-methylflavone (1), 5-hydroxy-7-methoxy-2-(4-methoxyphenyl)chromen-4-one, which displayed considerable activity against Mtb strain mc6230 in both log phase growth and metabolically downshifted hypoxic cultures.
CONCLUSIONS
The present study demonstrated that an O-methylflavone constituent of Artemisia afra explains part of the activity of this plant against Mtb. This result contributes to a mechanistic understanding of the reported anti-tubercular activity of A. afra and highlights the need for further study of this traditional medicinal plant and its active compounds.
PubMed: 38944359
DOI: 10.1016/j.jep.2024.118500 -
Cellular Signalling Jun 2024Circular RNAs (circRNAs) play a critical role in pathological mechanisms of Mycobacterium tuberculosis (Mtb) and can be used as a new biomarker for active tuberculosis...
Circular RNAs (circRNAs) play a critical role in pathological mechanisms of Mycobacterium tuberculosis (Mtb) and can be used as a new biomarker for active tuberculosis (ATB) diagnosis. Therefore, we identified significantly dysregulated circRNAs in ATB patients and healthy controls (HC) and explored their molecular mechanism. We found that hsa_circ_0002371 was significantly up-regulated in PBMCs of ATB patients and Mycobacterium tuberculosis H37Rv- or Mycobacterium bovis bacillus Calmette Guerin (BCG)-infected THP-1 cells. Functional experiments demonstrated that hsa_circ_0002371 inhibited autophagy in BCG-infected THP-1 cells and promoted intracellular BCG survival rate. In terms of mechanism, hsa_circ_0002371 facilitated the expression of hsa-miR-502-5p, as shown by bioinformatics and dual-luciferase reporter gene analysis, respectively. Notably, hsa-miR-502-5p inhibited autophagy via suppressing autophagy related 16 like 1 (ATG16L1) in BCG-infected macrophages and thus promoting intracellular BCG growth. In summation, hsa_circ_0002371 increased the suppression of hsa-miR-502-5p on ATG16L1 and inhibited autophagy to promote Mtb growth in macrophages. In Conclusion, our data suggested that hsa_circ_0002371 was significantly up-regulated in the PBMCs of ATB patients compared with HC. The hsa_circ_0002371/hsa-miR-502-5p/ATG16L1 axis promoted the survival of intracellular Mtb and inhibited autophagy in macrophages. Our findings suggested hsa_circ_0002371 could act as a potential diagnostic biomarker and therapeutic target.
PubMed: 38944259
DOI: 10.1016/j.cellsig.2024.111271 -
The Journal of Antimicrobial... Jun 2024Mycobacterium abscessus has emerged as an opportunistic pathogen responsible for lung infections, especially in cystic fibrosis patients. In spite of the production of...
OBJECTIVES
Mycobacterium abscessus has emerged as an opportunistic pathogen responsible for lung infections, especially in cystic fibrosis patients. In spite of the production of the broad-spectrum β-lactamase BlaMab, the carbapenem imipenem is recommended in the initial phase of the treatment of pulmonary infections. Here, we determine whether the addition of vaborbactam, a second-generation β-lactamase inhibitor belonging to the boronate family, improves the activity of β-lactams against M. abscessus.
METHODS
The activity of β-lactams, alone or in combination with vaborbactam, was evaluated against M. abscessus CIP104536 by determining MICs, time-killing and intramacrophage activity. Kinetic parameters for the inhibition of BlaMab by vaborbactam were determined by spectrophotometry.
RESULTS
The combination of vaborbactam (8 mg/L) with β-lactams decreased more than 8 times the MIC of amoxicillin (from >1024 to 128 mg/L) and 2 times the MICs of meropenem (from 16 to 8 mg/L) and imipenem (from 4 to 2 mg/L). The reduction of the MICs was less than that obtained with avibactam at 4 mg/L for amoxicillin (from >1024 to 16 mg/L, more than 64 times less) and for meropenem (from 16 to 4 mg/L, 4 times less). In vitro and intracellularly, M. abscessus was not killed by the meropenem/vaborbactam combination, in spite of significant in vitro inhibition of BlaMab by vaborbactam.
CONCLUSIONS
Inhibition of BlaMab by vaborbactam decreases the MIC of β-lactams, including that of meropenem. As meropenem/vaborbactam is clinically available, this combination offers an alternative therapeutic option that should be evaluated for the treatment of pulmonary infections due to M. abscessus.
PubMed: 38943535
DOI: 10.1093/jac/dkae181 -
Scientific Reports Jun 2024Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's Disease, a chronic granulomatous enteritis of ruminants. MAP establishes an...
Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's Disease, a chronic granulomatous enteritis of ruminants. MAP establishes an infection in the host via the small intestine. This requires the bacterium to adhere to, and be internalised by, cells of the intestinal tract. The effector molecules expressed by MAP for this purpose remain to be fully identified and understood. Mammalian cell entry (mce) proteins have been shown to enable other Mycobacterial species to attach to and invade host epithelial cells. Here, we have expressed Mce1A, Mce1D, Mce3C and Mce4A proteins derived from MAP on the surface of a non-invasive Escherichia coli to characterise their role in the initial interaction between MAP and the host. To this end, expression of mce1A was found to significantly increase the ability of the E. coli to attach and survive intracellularly in human monocyte-like THP-1 cells, whereas expression of mce1D was found to significantly increase attachment and invasion of E. coli to bovine epithelial cell-like MDBK cells, implying cell-type specificity. Furthermore, expression of Mce1A and Mce1D on the surface of a previously non-invasive E. coli enhanced the ability of the bacterium to infect 3D bovine basal-out enteroids. Together, our data contributes to our understanding of the effector molecules utilised by MAP in the initial interaction with the host, and may provide potential targets for therapeutic intervention.
Topics: Mycobacterium avium subsp. paratuberculosis; Paratuberculosis; Animals; Humans; Cattle; Bacterial Proteins; Bacterial Adhesion; Epithelial Cells; Escherichia coli; Cell Line; THP-1 Cells
PubMed: 38942800
DOI: 10.1038/s41598-024-65592-2