Subject(s)
Antitubercular Agents , Diarylquinolines , Microbial Sensitivity Tests , Mycobacterium tuberculosis , Reference Standards , Diarylquinolines/therapeutic use , Diarylquinolines/pharmacology , Humans , Mycobacterium tuberculosis/drug effects , Antitubercular Agents/pharmacology , Antitubercular Agents/therapeutic use , Tuberculosis, Multidrug-Resistant/drug therapy , Tuberculosis, Multidrug-Resistant/microbiologyABSTRACT
Implementation of whole genome sequencing (WGS) for patient care is hindered by limited Mycobacterium tuberculosis (Mtb) in clinical specimens and slow Mtb growth. We evaluated droplet multiple displacement amplification (dMDA) for amplification of minute amounts of Mtb DNA to enable WGS as an alternative to other Mtb enrichment methods. Purified genomic Mtb-DNA (0.1, 0.5, 1, and 5 pg) was encapsulated and amplified using the Samplix Xdrop-instrument and sequenced alongside a control sample using standard Illumina protocols followed by MAGMA-analysis. The control and 5 pg input dMDA samples underwent nanopore sequencing followed by Nanoseq and TB-profiler analysis. dMDA generated 105-2400 ng DNA from the 0.1-5 pg input DNA, respectively. Followed by Illumina WGS, dMDA raised mean sequencing depth from 7 × for 0.1 pg input DNA to ≥ 60 × for 5 pg input and the control sample. Bioinformatic analysis revealed a high number of false positive and false negative variants when amplifying ≤ 0.5 pg input DNA. Nanopore sequencing of the 5 pg dMDA sample presented excellent coverage depth, breadth, and accurate strain characterization, albeit elevated false positive and false negative variants compared to Illumina-sequenced dMDA sample with identical Mtb DNA input. dMDA coupled with Illumina WGS for samples with ≥ 5 pg purified Mtb DNA, equating to approximately 1000 copies of the Mtb genome, offers precision for drug resistance, phylogeny, and transmission insights.
Subject(s)
DNA, Bacterial , Genome, Bacterial , Mycobacterium tuberculosis , Whole Genome Sequencing , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/isolation & purification , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , Whole Genome Sequencing/methods , Humans , Nucleic Acid Amplification Techniques/methods , Nanopore Sequencing/methods , High-Throughput Nucleotide Sequencing/methods , Tuberculosis/microbiology , Tuberculosis/diagnosisABSTRACT
BACKGROUND: Non-tuberculous mycobacteria (NTM) infections are increasing in incidence and associated mortality. NTM are naturally resistant to a variety of antibiotics, complicating treatment. We conducted a literature assessment on the efficacy of bedaquiline in treating NTM species in vitro and in vivo (animal models and humans); meta-analyses were performed where possible. METHOD: Four databases were searched using specific terms. Publications were included according to predefined criteria. Bedaquiline's impact on NTM in vitro, MICs and epidemiological cut-off (ECOFF) values were evaluated. A meta-analysis of bedaquiline efficacy against NTM infections in animal models was performed. Culture conversion, cure and/or relapse-free cure were used to evaluate the efficacy of bedaquiline in treating NTM infection in humans. RESULTS: Fifty studies met the inclusion criteria: 33 assessed bedaquiline's impact on NTM in vitro, 9 in animal models and 8 in humans. Three studies assessed bedaquiline's efficacy both in vitro and in vivo. Due to data paucity, an ECOFF value of 0.5 mg/mL was estimated for Mycobacterium abscessus only. Meta-analysis of animal studies showed a 1.86× reduction in bacterial load in bedaquiline-treated versus no treatment within 30 days. In humans, bedaquiline-including regimens were effective in treating NTM extrapulmonary infection but not pulmonary infection. CONCLUSIONS: Bedaquiline demonstrated strong antibacterial activity against various NTM species and is a promising drug to treat NTM infections. However, data on the genomic mutations associated with bedaquiline resistance were scarce, preventing statistical analyses for most mutations and NTM species. Further studies are urgently needed to better inform treatment strategies.
Subject(s)
Mycobacterium Infections, Nontuberculous , Nontuberculous Mycobacteria , Humans , Mycobacterium Infections, Nontuberculous/drug therapy , Mycobacterium Infections, Nontuberculous/microbiology , Diarylquinolines/pharmacology , Diarylquinolines/therapeutic use , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic useABSTRACT
BACKGROUND: The genetic architecture of rheumatoid arthritis (RA) and osteoarthritis (OA) are still unclear. Although RA and OA have quite different causes, they share synovial inflammation, risk factors, and some disease-associated genes, including the integrin subunit ß2 (ITGB2)/CD18 gene involved in extracellular matrix interactions and immune cell signaling. However, the functional role of ITGB2 genetic variants, its circulating expression pattern, and their clinical usefulness in RA and OA remain unexplored. Our study appraised the association of ITGB2 rs2070946 single nucleotide polymorphism with the vulnerability to RA and OA and its influence on ITGB2 mRNA expression, along with the potential of serum ITGB2 expression in RA and OA diagnosis. METHODS: This study included 70 RA patients, 70 primary OA patients, and 60 healthy volunteers. Genotyping and gene expression analysis were performed using qPCR. Bioinformatics analysis was employed to construct the protein-protein interaction (PPI) network of ITGB2. RESULTS: Serum ITGB2 mRNA expression was upregulated in both RA and OA compared to healthy controls. ITGB2 rs2070946 was associated with escalating risk of both diseases. RA patients harboring the rs2070946 CC or TC + CC genotypes had higher serum ITGB2 expression than the TT genotype carriers. Likewise, OA patients having the minor homozygote CC genotype had higher serum ITGB2 expression than those carrying the TT, TC or TT + TC genotypes. Serum ITGB2 expression showed profound diagnostic potential for RA and OA in receiver-operating characteristic analysis. In RA, serum ITGB2 expression positively correlated with rheumatoid factor and disease activity score 28 (DAS28). The ITGB2-PPI network enriched in cell-cell adhesion, ICAM-3 receptor activity, T-cell activation, leukocyte adhesion, complement binding, and NF-κB, tumor necrosis factor, and interleukin signaling pathways. CONCLUSION: These findings embrace the impact of ITGB2 rs2070946 as a novel genetic biomarker of both RA and OA, which could alter the ITGB2 expression. Serum ITGB2 expression could aid in timely diagnosis of RA and OA.
Subject(s)
Arthritis, Rheumatoid , Osteoarthritis , Humans , CD18 Antigens/genetics , Egypt , Arthritis, Rheumatoid/genetics , Osteoarthritis/genetics , Polymorphism, Single Nucleotide , RNA, MessengerABSTRACT
Mycobacterium tuberculosis whole-genome sequencing (WGS) is a powerful tool as it can provide data on population diversity, drug resistance, disease transmission, and mixed infections. Successful WGS is still reliant on high concentrations of DNA obtained through M. tuberculosis culture. Microfluidics technology plays a valuable role in single-cell research but has not yet been assessed as a bacterial enrichment strategy for culture-free WGS of M. tuberculosis. In a proof-of-principle study, we evaluated the use of Capture-XT, a microfluidic lab-on-chip cleanup and pathogen concentration platform to enrich M. tuberculosis bacilli from clinical sputum specimens for downstream DNA extraction and WGS. Three of the four (75%) samples processed by the microfluidics application passed the library preparation quality control, compared to only one of the four (25%) samples not enriched by the microfluidics M. tuberculosis capture application. WGS data were of sufficient quality, with mapping depth of ≥25× and 9 to 27% of reads mapping to the reference genome. These results suggest that microfluidics-based M. tuberculosis cell capture might be a promising method for M. tuberculosis enrichment in clinical sputum samples, which could facilitate culture-free M. tuberculosis WGS. IMPORTANCE Diagnosis of tuberculosis is effective using molecular methods; however, a comprehensive characterization of the resistance profile of Mycobacterium tuberculosis often requires culturing and phenotypic drug susceptibility testing or culturing followed by whole-genome sequencing (WGS). The phenotypic route can take anywhere from 1 to >3 months to result, by which point the patient may have acquired additional drug resistance. The WGS route is a very attractive option; however, culturing is the rate-limiting step. In this original article, we provide proof-of-principle evidence that microfluidics-based cell capture can be used on high-bacillary-load clinical samples for culture-free WGS.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis , Humans , Mycobacterium tuberculosis/genetics , Microfluidics , Microbial Sensitivity Tests , Tuberculosis/microbiology , Whole Genome Sequencing , Antitubercular Agents/pharmacologyABSTRACT
Whole genome sequencing (WGS) can investigate the entire Mycobacterium tuberculosis (Mtb) genome but currently requires large amounts of mycobacterial DNA, necessitating culture. Culture-free Mtb WGS could revolutionize the clinical use of WGS but is hampered by the high viscosity, low mycobacterial load, and high contamination with bacterial and human DNA in sputum samples. To improve the sputum liquefaction and decontamination step prior to DNA extraction, we assessed the efficiency of Myco-TB, MycoPrep, and Sputolysin with/without TiKa-Kic in liquefying and decontaminating sputum and aimed to evaluate the effect of these approaches on mycobacterial viability, and Mtb DNA quality and quantity. Experiments using spiked sputum samples showed that Myco-TB and BD MycoPrep with standard (15 min) or increased (30 min) incubation time, but not reduced (7,5 min) incubation time performed well in liquefying and decontaminating sputum. No difference in DNA quality or quantity, contamination, or the amount of human DNA present was observed. In comparison, Sputolysin with/without TiKa-Kic was less effective for liquefaction and decontamination of sputum. PCR amplification of the human GAPDH gene after sputum treatment, showed the presence of human DNA in all samples, regardless of sputum treatment. Focused efforts are needed to deplete contaminating DNA for culture-free Mtb WGS.
Subject(s)
Decontamination , Mycobacterium tuberculosis , Specimen Handling , Sputum , Humans , Bacteriological Techniques/methods , Decontamination/methods , Decontamination/standards , Decontamination/statistics & numerical data , Mycobacterium tuberculosis/isolation & purification , Mycobacterium tuberculosis/metabolism , Sensitivity and Specificity , Specimen Handling/methods , Specimen Handling/standards , Sputum/microbiology , Tuberculosis/diagnosisABSTRACT
BACKGROUND: Bedaquiline is a crucial drug for control of rifampicin-resistant tuberculosis. Molecular drug resistance assays could facilitate effective use of bedaquiline and surveillance of drug resistance emergence. To facilitate molecular assay development, we aimed to identify genomic markers of bedaquiline resistance. METHODS: In this systematic review and individual isolate analysis, we searched Europe PubMed Central and Scopus for studies published from the inception of each database until Oct 19, 2020, that assessed genotypic and phenotypic bedaquiline resistance in clinical or non-clinical Mycobacterium tuberculosis isolates. All studies reporting on the assessment of variants in the four genes of interest (Rv0678, atpE, pepQ, and Rv1979c) and phenotypic bedaquiline data in both clinical and non-clinical samples were included. We collated individual isolate data from eligible studies to assess the association between genomic variants with phenotypic bedaquiline resistance, using a standardised method endorsed by WHO. Risk of bias of the extracted data was independently assessed by two authors using the Quality Assessment of Diagnostic Accuracy Studies tool for clinical studies and Systematic Review Center for Laboratory Animal Experimentation tool for animal studies. The primary outcome was to identify mutations associated with resistance in four genes of interest (Rv0678, atpE, pepQ, and Rv1979c); for each genomic variant, the odds ratio (OR), 95% CI, and p value were calculated to identify resistance markers associated with bedaquiline resistance. This study is registered with PROSPERO, CRD42020221498. FINDINGS: Of 1367 studies identified, 41 published between 2007 and 2020 were eligible for inclusion. We extracted data on 1708 isolates: 1569 (91·9%) clinical isolates and 139 (8·1%) non-clinical isolates. We identified 237 unique variants in Rv0678, 14 in atpE, 28 in pepQ, and 11 in Rv1979c. Most clinical isolates with a single variant reported in Rv0678 (229 [79%] of 287 variants), atpE (14 [88%] of 16 variants), pepQ (32 [100%] of 32 variants), or Rv1979c (115 [98%] of 119 variants) were phenotypically susceptible to bedaquiline. Except for the atpE 187GâC (OR ∞, [95% CI 13·28-∞]; p<0·0001) and Rv0678 138_139insG (OR 6·91 [95% CI 1·16-47·38]; p=0·016) variants, phenotypic-genotypic associations were not significant (p≥0·05) for any single variant in Rv0678, atpE, pepQ, and Rv1979c. INTERPRETATION: Absence of clear genotypic-phenotypic associations for bedaquiline complicates the development of molecular drug susceptibility tests. A concerted global effort is urgently needed to assess the genotypic and phenotypic drug susceptibility of M tuberculosis isolates, especially in patients who have received unsuccessful bedaquiline-containing regimens. Treatment regimens should be designed to prevent emergence of bedaquiline resistance and phenotypic drug susceptibility tests should be used to guide and monitor treatment. FUNDING: Research Foundation Flanders, South African Medical Research Council, Department of Science and Innovation - National Research Foundation, National Institute of Health Institute of Allergy and Infectious Diseases, and Doris Duke Charitable Foundation.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis, Lymph Node , Tuberculosis, Multidrug-Resistant , Animals , Antitubercular Agents/pharmacology , Data Analysis , Diarylquinolines , Humans , Microbial Sensitivity Tests , Mycobacterium tuberculosis/genetics , Tuberculosis, Lymph Node/drug therapy , Tuberculosis, Multidrug-Resistant/drug therapyABSTRACT
BACKGROUND: Whole genome sequencing (WGS) is increasingly used for Mycobacterium tuberculosis (Mtb) research. Countries with the highest tuberculosis (TB) burden face important challenges to integrate WGS into surveillance and research. METHODS: We assessed the global status of Mtb WGS and developed a 3-week training course coupled with long-term mentoring and WGS infrastructure building. Training focused on genome sequencing, bioinformatics and development of a locally relevant WGS research project. The aim of the long-term mentoring was to support trainees in project implementation and funding acquisition. The focus of WGS infrastructure building was on the DNA extraction process and bioinformatics. FINDINGS: Compared to their TB burden, Asia and Africa are grossly underrepresented in Mtb WGS research. Challenges faced resulted in adaptations to the training, mentoring and infrastructure building. Out-of-date laptop hardware and operating systems were overcome by using online tools and a Galaxy WGS analysis pipeline. A case studies approach created a safe atmosphere for students to formulate and defend opinions. Because quality DNA extraction is paramount for WGS, a biosafety level 3 and general laboratory skill training session were added, use of commercial DNA extraction kits was introduced and a 2-week training in a highly equipped laboratory was combined with a 1-week training in the local setting. INTERPRETATION: By developing and sharing the components of and experiences with a sequencing and bioinformatics training program, we hope to stimulate capacity building programs for Mtb WGS and empower high-burden countries to play an important role in WGS-based TB surveillance and research.
Subject(s)
Computational Biology , Genome, Bacterial , Mycobacterium tuberculosis/genetics , Tuberculosis/genetics , Whole Genome Sequencing , Africa/epidemiology , Asia/epidemiology , Cost of Illness , Humans , Mycobacterium tuberculosis/isolation & purification , Tuberculosis/epidemiologyABSTRACT
Tuberculosis claims more human lives than any other bacterial infectious disease and represents a clear and present danger to global health as new tools for vaccination, treatment, and interruption of transmission have been slow to emerge. Additionally, tuberculosis presents with notable clinical heterogeneity, which complicates diagnosis, treatment, and the establishment of nonrelapsing cure. How this heterogeneity is driven by the diversity ofclinical isolates of the causative agent, Mycobacterium tuberculosis, has recently garnered attention. Herein, we review advances in the understanding of how naturally occurring variation in clinical isolates affects transmissibility, pathogenesis, immune modulation, and drug resistance. We also summarize how specific changes in transcriptional responses can modulate infection or disease outcome, together with strain-specific effects on gene essentiality. Further understanding of how this diversity of M. tuberculosis isolates affects disease and treatment outcomes will enable the development of more effective therapeutic options and vaccines for this dreaded disease.
Subject(s)
Genetic Variation/genetics , Mycobacterium tuberculosis/genetics , Animals , Genotype , Humans , Transcription, Genetic/genetics , Tuberculosis/microbiologyABSTRACT
Ageing is an unavoidable, universal, biological phenomenon affecting all organisms, which involves variable declines of individuals motor and memory capabilities. This study aimed to investigate the potential ameliorating effects of curcumin C3 complex, Astragalus membranaceus and blueberry on certain age-related biochemical alterations in rat liver. Four groups of rats, aged 12 months-old, were used. The first group; aged control group in which rats were left without any treatment until the age of 17 months. The other three groups received daily by oral gavage for 5 months the following supplements; curcumin C3 complex (110 mg/kg), Astragalus membranaceus (100 mg/kg) and blueberry (100 mg/kg) respectively. Additionally, a fifth group of rats, aged 5 months-old, was used as an adult control group. Our supplements alleviated ageing-induced redox state imbalance and inflammation as evidenced by reduction of hepatic thiobarbituric acid reactive substances and 8-hydroxydeoxyguanosine levels, restoration of total antioxidant capacity and nitric oxide contents, and lessening of lipofuscin deposition. All supplements decreased hepatic interlukin-6 gene expression and serum levels. Notably, Astragalus membranaceus and blueberry upregulated hepatic telomerase reverse transcriptase gene expression and increased telomere length. Our findings recommend the use of these natural hepatoprotective supplements for the elderly to promote healthy ageing and minimize the risk of age-related liver diseases.
Subject(s)
Blueberry Plants , Curcumin , Liver Diseases , Animals , Astragalus propinquus , Curcumin/pharmacology , RatsABSTRACT
Bedaquiline resistance within Mycobacterium tuberculosis may arise through efflux-based (rv0678) or target-based (atpE) pathway mutations. M. tuberculosis mutant populations from each of five sequential steps in a passaging approach, using a pyrazinamide-resistant ATCC strain, were subjected to MIC determinations and whole-genome sequencing. Exposure to increasing bedaquiline concentrations resulted in increasing phenotypic resistance (up to >2 µg/ml) through MIC determination on solid medium (Middlebrook 7H10). rv0678 mutations were dynamic, while atpE mutations were fixed, once occurring. We present the following hypothesis for in vitro emergence of bedaquiline resistance: rv0678 mutations may be the first transient step in low-level resistance acquisition, followed by high-level resistance due to fixed atpE mutations.
Subject(s)
Bacterial Proteins/genetics , Diarylquinolines/pharmacology , Drug Resistance, Bacterial/drug effects , Mutation , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Antitubercular Agents/pharmacology , Bacterial Proton-Translocating ATPases/genetics , Drug Resistance, Bacterial/genetics , Microbial Sensitivity TestsABSTRACT
South Africa remains challenged with a high tuberculosis burden accompanied by an increase in drug resistant cases. We assessed the use of the Illumina MiSeq, a next-generation sequencing platform for whole genome sequencing, followed by bioinformatic analysis using a commercial software package to determine resistance to selected drugs used for Mycobacterium tuberculosis treatment in our setting. Whole genome sequencing shows potential as a diagnostic platform for the detection of drug resistance in Mycobacterium tuberculosis with the provision of information for several drugs simultaneously.
ABSTRACT
Six in vitro clofazimine-resistant spontaneous mutants obtained from a wild-type or pyrazinamide-resistant ATCC reference strain were selected to evaluate bedaquiline cross-resistance. The reverse was conducted for bedaquiline mutants. All clofazimine mutants harboring an rv0678 mutation displayed phenotypic cross-resistance. We observed the same for rv0678 bedaquiline mutants; however, atpE bedaquiline mutants showed no phenotypic cross-resistance. This confirms that upfront clofazimine usage may impact subsequent bedaquiline use due to a shared efflux resistance pathway.
Subject(s)
Antitubercular Agents/pharmacology , Clofazimine/pharmacology , Diarylquinolines/pharmacology , Membrane Transport Proteins/genetics , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Drug Resistance, Multiple, Bacterial/genetics , Microbial Sensitivity Tests , Tuberculosis, Multidrug-Resistant/drug therapy , Tuberculosis, Multidrug-Resistant/geneticsABSTRACT
OBJECTIVES: To assess the performance of an innovative method of transporting sputum to centralised facilities for molecular detection of Mycobacterium tuberculosis: using a swab to inoculate sputum in a transport medium, PrimeStore(®) Molecular Transport Medium (PS-MTM). METHODS: Two sputum specimens were obtained from suspected patients with tuberculosis (TB) at rural healthcare facilities in South Africa. A swab was taken from each specimen and placed into PS-MTM, prior to it being processed by either liquid culture or Xpert MTB/RIF assay (Xpert). RESULTS: A total of 141 patients (including 47 with laboratory-confirmed TB) were included in this analysis. M. tuberculosis was detected at 29% by culture and 29% by Xpert, whereas 31% tested positive by IS6110 real-time PCR of PS-MTM from the culture and 36% from the Xpert-paired specimen. Concordance between the method under evaluation with culture was 82% (McNemar, P = 0.55) and 84% (McNemar, P = 0.05) for Xpert. Stratified by culture result, the detection rate by IS6110 real-time PCR of PS-MTM was similar to Xpert for patients with positive culture (P = 0.32), but significantly higher if culture was negative (P = 0.008). CONCLUSIONS: These results suggest that swab collection of sputum into PS-MTM for transport is a promising method for diagnosis of TB in rural healthcare settings, thereby potentially improving the options available for molecular diagnosis of TB in countries incapable of applying decentralised high-tech molecular testing.
Subject(s)
Culture Media , Mycobacterium tuberculosis , Rural Population , Specimen Handling/methods , Sputum/microbiology , Transportation , Tuberculosis, Pulmonary/microbiology , Adult , Female , Humans , Male , Mycobacterium tuberculosis/genetics , Real-Time Polymerase Chain Reaction , Rifampin , South AfricaABSTRACT
Chronic hepatitis is recognized as a worldwide health problem that gradually progresses towards cirrhosis and hepatocellular carcinoma. Despite the large number of experiments using animal models for allergic hepatitis, it is still difficult to produce a picture of chronic hepatitis. Therefore, this study was conducted to introduce an animal model approximating to the mechanism of chronicity in human hepatitis. The study also aimed to examine the hepatoprotective effects of curcumin, silybin phytosome(®) and α-R-lipoic acid against thioacetamide (TAA)-induced chronic hepatitis in rat model. TAA was administered intraperitoneally at a dose of 200 mg/kg three times weekly for 4 weeks. At the end of this period, a group of rats was killed to assess the development of chronic hepatitis in comparison with their respective control group. TAA administration was then discontinued, and the remaining animals were subsequently allocated into four groups. Group 1 was left untreated, whereas groups 2-4 were allowed to receive daily oral doses of curcumin, silybin phytosome(®) or α-R-lipoic acid, respectively, for 7 weeks. Increases in hepatic levels of malondialdehyde associated with TAA administration were inhibited in groups receiving supplements. Furthermore, glutathione depletion, collagen deposition, macrophage activation and nuclear factor κappa-B expression as well as tumour necrosis factor-α and interleukin-6 levels were significantly decreased in response to supplements administration. Serological analysis of liver function and liver histopathological examination reinforced the results. The above evidence collectively indicates that the antioxidant and anti-inflammatory activities of curcumin, silybin phytosome(®) and α-R-lipoic acid may confer therapeutic efficacy against chronic hepatitis.
Subject(s)
Curcumin/pharmacology , Hepatitis, Chronic/drug therapy , Silymarin/analogs & derivatives , Thioctic Acid/pharmacology , Administration, Oral , Animals , Anti-Inflammatory Agents/administration & dosage , Anti-Inflammatory Agents/pharmacology , Antioxidants/administration & dosage , Antioxidants/pharmacology , Curcumin/administration & dosage , Cytokines/metabolism , Disease Models, Animal , Hepatitis, Chronic/pathology , Male , Oxidative Stress/drug effects , Rats , Rats, Wistar , Silybin , Silymarin/administration & dosage , Silymarin/pharmacology , Thioacetamide/toxicity , Thioctic Acid/administration & dosageABSTRACT
BACKGROUND: Modern molecular-based approaches for the detection of Mycobacterium tuberculosis in sputum samples promise quicker and more accurate detection of cases. However, processing sputum samples at central diagnostic facilities provides a diagnostic approach, but requires a safe and efficient system that is not affected by transport delays and ambient temperature to be feasible. We evaluated the technical properties of PrimeStore®-Molecular Transport Medium (PS-MTM) for its ability to inactivate mycobacteria, ensuring stability of DNA over time at ambient temperatures and to assess the compatibility of the transport medium with DNA extraction systems. METHODS: Assessment of the transport medium for application of sputum samples processed for the detection of M. tuberculosis included the inactivation of M. tuberculosis in spiked sputum samples, compatibility of the medium with three commercial nucleic extraction systems and stability of DNA in the medium at ambient temperature over 28 days. We further performed a clinical laboratory evaluation on 256 sputum specimens sent for tuberculosis investigation. RESULTS: Complete inactivation of M. tuberculosis occurred within 30 min of exposure at a ratio of 1:3 for sputum to PS-MTM. Sputum specimen in PS-MTM showed very good compatibility with automated bead-based extraction systems, producing high DNA output (estimated lower limits of detection: ~170 CFU/ml). Furthermore, PS-MTM samples remained stable over 28 days at ambient temperature displaying no significant change over time in Ct-values (<5% on a mean starting value of 22.47). Of the 256 clinical sputum specimens, 10.2% were culture positive and 11.0% were positive by real-time PCR of PS-MTM samples. CONCLUSIONS: Collecting and transporting sputum from TB suspects in PS-MTM offer safe transport at ambient temperature, DNA stability for extended periods without cooling and specimens directly suitable for molecular testing. This novel approach may support introduction and further scale-up of molecular diagnostics for TB in resource-limited settings.
Subject(s)
Culture Media/pharmacology , Molecular Typing/methods , Mycobacterium tuberculosis/drug effects , Specimen Handling/methods , Sputum/microbiology , Culture Media/chemistry , Humans , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/isolation & purification , Tuberculosis/diagnosis , Tuberculosis/microbiologyABSTRACT
Liver cirrhosis is the final consequence of a progressive fibrotic process characterized by excessive collagen deposition and destruction of the normal liver architecture. This study aimed to investigate the protective effects of curcumin, silybin-phytosome and alpha-R-lipoic acid against thioacetamide-induced cirrhosis. Male rats were allocated into five groups of which one group received saline and served as normal control. Animals from groups 2-5 were treated with thioacetamide administered intraperitoneally at a dose of 200 mg/kg 3 times per week for 7 weeks. Group 2 was left untreated while groups from 3 to 5 were given a daily oral dose of curcumin, silybin-phytosome or alpha-R-lipoic acid simultaneously with thioacetamide. Increases in hepatic levels of malondialdehyde (MDA) and protein carbonyls (Pr Co) associated with thioacetamide administration were partially blocked in those groups receiving supplements. Glutathione (GSH) depletion, collagen deposition, matrix metalloproteinase-2 (MMP-2) activity, transforming growth factor-ß1 (TGF-ß1) level as well as α-smooth muscle actin (α-SMA) and heat shock protein-47 (HSP-47) gene expressions were also decreased in response to supplements administration. Serological analysis of liver function and histopathological examination reinforced the results. In conclusion, the present study highlights the antioxidant and the antifibrotic potentials of these supplements against chronic liver diseases caused by ongoing hepatic damage.
Subject(s)
Antioxidants/therapeutic use , Curcumin/therapeutic use , Liver Cirrhosis/chemically induced , Silymarin/analogs & derivatives , Thioacetamide/toxicity , Thioctic Acid/therapeutic use , Animals , Biomarkers , Chemical and Drug Induced Liver Injury/prevention & control , Gene Expression Regulation , HSP47 Heat-Shock Proteins/genetics , HSP47 Heat-Shock Proteins/metabolism , Liver Cirrhosis/drug therapy , Liver Cirrhosis/pathology , Male , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 2/metabolism , Oxidative Stress/drug effects , Rats , Rats, Wistar , Silybin , Silymarin/therapeutic use , Transforming Growth Factor beta1/genetics , Transforming Growth Factor beta1/metabolismABSTRACT
The free radical theory of ageing postulates that age-associated neurodegeneration is caused by an imbalance between pro-oxidants and antioxidants resulting in oxidative stress. The current study showed regional variation in brain susceptibility to age-associated oxidative stress as shown by increased lipofuscin deposition and protein carbonyl levels in male rats of age 15-16 months compared to control ones (3-5 months). The hippocampus is the area most vulnerable to change compared to the cortex and cerebellum. However, proteasomal enzyme activity was not affected by age in any of the brain regions studied. Treatment with melatonin or coenzyme Q10 for 4 weeks reduced the lipofuscin content of the hippocampus and carbonyl level. However, both melatonin and coenzyme Q10 treatments inhibited beta-glutamyl peptide hydrolase activity. This suggests that these molecules can alter proteasome function independently of their antioxidant actions.