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1.
Talanta ; 236: 122902, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34635273

RESUMO

Rapid diagnosis of tuberculosis disease (TB) still remained a pressing need for TB control efforts all over the world. However, the existing detection approaches cannot satisfy demand of rapid detection of clinical Mycobacterium tuberculosis (M. tuberculosis) because of the long detection time and high cost. Herein, we proposed a new M. tuberculosis piezoelectric sensor based on AuNPs-mediated enzyme assisted signal amplification. A hairpin-shaped DNA duplex with a protrusion of the 3' end was designed. In the presence of specific 16 S rDNA fragment of M. tuberculosis, the hairpin probe was opened, which triggered the selective cleavage of hairpin probe by Exonuclease III (Exo III), resulting in the release of uncut DNA probe and target DNA. The released target DNA hybridized with another hairpin-shaped DNA duplex, and a new digestion cycle was started, thus generating large amounts of uncut DNA probes. The uncut DNA was pulled to the electrode surface by the hybridization with capture probe modified on the electrode. Subsequently detection probe labeled AuNPs was hybridized with uncut DNA and entered between the two electrodes. The AuNPs linked to hybridized detection probe were grown in the HAuCl4 and Nicotinamide adenine dinucleotide (NADH) solution and offered the conductive connection between the gaps of electrode. The changes were monitored by the piezoelectric sensor. The piezoelectric biosensor could achieve a detection of M. tuberculosis (102-108 CFU mL-1) within 3 h, the detection limit (LOD) was 30 CFU mL-1. The methodology could be transformed into different microbial targets, which is suitable for further development of small portable equipment and multifunctional detection.


Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , Mycobacterium tuberculosis , DNA Ribossômico , Ouro , Mycobacterium tuberculosis/genética
2.
Biosens Bioelectron ; 195: 113663, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34610534

RESUMO

Tuberculosis (TB) remains the high-risk infectious pathogen that caused global pandemic and high mortality, particularly in the areas lack in health resources. Clinical TB screening and diagnosis are so far mainly conducted on limited types of commercial platforms, which are expensive and require skilled personnel. In this work, we introduced a low-cost and portable finger-driven microfluidic chip (named Fd-MC) based on recombinase polymerase amplification (RPA) for rapid on-site detection of TB. After injection of the pre-treated sample solution, the pre-packaged buffer was driven by the pressure generated by the finger-actuated operation to accomplish sequential processes of diagnosis in a fully isolated microchannel. An in-situ fluorescence strategy based on FAM-probe was implemented for on-chip results read-out though a hand-held UV lamp. Hence, the Fd-MC proved unique advantageous for avoiding the risk of infection or environmental contamination. In addition, the Fd-MC was designed for multiplexed detection, which is able to not only identify TB/non-TB infection, but also differentiate between human Mycobacterium tuberculosis and Mycobacterium bovis. The platform was verified in 37 clinical samples, statistically with 100% specificity and 95.2% sensitivity as compared to commercial real-time RPA. Overall, the proposed platform eliminates the need on external pumps and skilled personnel, holding promise to POC testing in the resource-limited area.


Assuntos
Técnicas Biossensoriais , Mycobacterium bovis , Mycobacterium tuberculosis , Tuberculose , Humanos , Mycobacterium tuberculosis/genética , Sistemas Automatizados de Assistência Junto ao Leito , Tuberculose/diagnóstico
3.
Front Cell Infect Microbiol ; 11: 756854, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34765568

RESUMO

Antigen-specific gamma-delta (γδ) T cells are important in exhibiting anti-mycobacterial immunity, but their role in latent tuberculosis (LTB) with diabetes mellitus (DM) or pre-DM (PDM) and non-DM comorbidities have not been studied. Thus, we have studied the baseline, mycobacterial (PPD, WCL), and positive control antigen-stimulated γδ T cells expressing Th1 (IFNγ, TNFα, IL-2) and Th17 (IL-17A, IL-17F, IL-22) cytokine as well as cytotoxic (perforin [PFN], granzyme [GZE B], granulysin [GNLSN]) and immune (GMCSF, PD-1, CD69) markers in LTB (DM, PDM, NDM) comorbidities by flow cytometry. In the unstimulated (UNS) condition, we did not observe any significant difference in the frequencies of γδ T cells expressing Th1 and Th17 cytokine, cytotoxic, and immune markers. In contrast, upon PPD antigen stimulation, the frequencies of γδ T cells expressing Th1 (IFNγ, TNFα) and Th17 (IL-17F, IL-22) cytokine, cytotoxic (PFN, GZE B, GNLSN), and immune (CD69) markers were significantly diminished in LTB DM and/or PDM individuals compared to LTB NDM individuals. Similarly, upon WCL antigen stimulation, the frequencies of γδ T cells expressing Th1 (TNFα) and Th17 (IL-17A, IL-22) cytokine, cytotoxic (PFN), and immune (PD-1, CD69) markers were significantly diminished in LTB DM and/or PDM individuals compared to LTB NDM individuals. Finally, upon P/I stimulation we did not observe any significant difference in the γδ T cell frequencies expressing cytokine, cytotoxic, and immune markers between the study populations. The culture supernatant levels of IFNγ, TNFα, and IL-17A cytokines were significantly increased in LTB DM and PDM after stimulation with Mtb antigens compared to LTB NDM individuals. Therefore, diminished γδ T cells expressing cytokine, cytotoxic, and other immune markers and elevated levels of cytokines in the supernatants is a characteristic feature of LTB PDM/DM co-morbidities.


Assuntos
Tuberculose Latente , Mycobacterium tuberculosis , Estado Pré-Diabético , Biomarcadores , Comorbidade , Citocinas , Humanos , Células Th17
5.
BMC Infect Dis ; 21(1): 1175, 2021 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-34809602

RESUMO

INTRODUCTION: Globally anti-tuberculosis drug resistance is one of the major challenges affecting control and prevention of tuberculosis. Kenya is ranked among 30 high burden TB countries globally. However, there is scanty information on second line antituberculosis drug resistance among tuberculosis patients. Therefore, this study aimed at determining Mycobacterium tuberculosis drug resistant strain distribution pattern in 10 counties of Western Kenya among HIV positive and negative patients. METHOD: A cross-sectional study was conducted in Western Kenya, which comprises 10 counties. A multistage sampling method was used where a single sub-county was randomly selected followed by sampling one high volume health facility from each sub-county. Consenting study subjects with at least two smear positive sputum at the time of enrolment were randomly selected. The collected sputum was decontaminated with N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) and then stained with Ziehl Neelsen Stain before visualizing the presence of bacilli under microscope at ×100 magnification with oil immersion. Further, the identified bacilli were cultured and susceptibility test carried out using known first and second line antimycobacterial tuberculosis. HIV testing was carried out using Determine® HIV-1/2 rapid test (Abbot Diagnostics, Maidenhead, United Kingdom). Those who had smear converted were dropped from the study. Finally, drug susceptibility pattern across the 10 counties of Western Kenya was evaluated. RESULTS: Our study showed that Mycobacterium tuberculosis drug resistance among HIV negative and positive cases in Western Kenya was prevalent in all the 10 counties surveyed. Based on the drug susceptibility tests, 53.2% and 42.7% of the study samples were resistant to at least one antituberculosis drug among HIV negative and HIV positive patients respectively. The data analysis revealed that among the HIV-positive and HIV-negative patients, resistance to INH was predominant (28.5%, and 23.6%, respectively), followed by RIF (16.4% and 14.6% respectively). Second-line drug resistant strains identified among HIV negative patients included Ethionamide (0.3%), Gatifloxacin (0.3%), Amikacin (0.3%) and Capreomycin (0.3%). There was no second line drug monoresistance among HIV positive TB patients. Multi/poly drug resistance were noted among HIV-negative patients in, INH + AMK (0.7%), INH + PZA (1%), INH + GFX (0.7%, INH + ETO (0.7%, STY + ETO (1%), ETH + ETO (1.0%), INH + KAN (0.7%) and INH + CAP (0.7%) strains/cases at 95% confidence interval. Among HIV positive patients INH + GFX (1.1%), INH + ETO (0.4%) and INH + KAN (0.4%) strains of M. tuberculosis were identified with a confidence interval of 95%. Geographical distribution patterns analysis of M. tuberculosis drug polyresistant strains across the 10 counties were recorded. Among HIV TB patients, resistant strains were identified in Nyamira (INH + GFX, INH + KAN), Bungoma ((ETO + STY), Busia (ETH + ETO and STY + ETO) Homabay (RIF + AMK. ETO + ETH and ETO + STY), Kisumu (ETH + ETO and PZA + ETO) and in Kakamega, Kisii and Vihiga (INH + KAN and RIF + AMK). There was no M. tuberculosis polyresistant strain identified in Migori and Siaya counties. Among HIV positive TB patients, M. tuberculosis resistant strains were identified in three counties, Nyamira (INH + KAN) Homabay (INH + GFX and INH + AMK) and Kakamega (INH + GFX). There was no polyresistant M. tuberculosis strain identified in Migori, Bungoma, Kisii, Vihiga, Busia, Siaya and Kisumu Counties. DISCUSSION: The distribution patterns of M. tuberculosis drug resistance among HIV negative and positive TB patients could be as a result of reported high prevalence of HIV in Western Kenya counties especially the area under study. Tuberculosis is one of the opportunistic diseases that have been shown to be the major cause of AIDS among HIV infected patients. Resent reports by National AIDS Control Council shows that Kisumu, Siaya, Homabay, Migori, Busia have the overall leading in HIV prevalence in Kenya. The low prevalence of drug resistant strains among HIV tuberculosis patients could be as a result of drug adherence attitude adopted by HIV patients, availability of continuous counselling and close follow up and notification by healthcare workers and community health volunteers. CONCLUSION: Drug resistant M. tuberculosis strains prevalence is still high among HIV negative and positive patients in Western Kenya with the most affected being HIV negative TB patients. It is therefore probable that the existing control measures are not adequate to control transmission of drug resistant strains. Further, miss diagnosis or delayed diagnosis of TB patients could be contributing to the emergence of M. tuberculosis drug polyresistant strains. RECOMMENDATION: Based on the result of this study, regular TB drug resistance surveillance should be conducted to ensure targeted interventions aimed at controlling increased transmission of the tuberculosis drug resistant strains among HIV/AIDS and HIV negative patients. There is also need for improved drug resistant infection control measures, timely and rapid diagnosis and enhanced and active screening strategies of tuberculosis among suspected TB patients need to be put in place. Further, studies using a larger patient cohort and from counties across the country would shed much needed insights on the true national prevalence of different variants of M. tuberculosis drug resistance.


Assuntos
Infecções por HIV , Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Estudos Transversais , Resistência a Medicamentos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/epidemiologia , Humanos , Quênia/epidemiologia , Testes de Sensibilidade Microbiana , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia
6.
BMC Res Notes ; 14(1): 413, 2021 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-34776013

RESUMO

OBJECTIVE: To determine at two distinct time points the prevalence of resistance to ofloxacin (OFX), the representative class drug of fluoroquinolones (FQs), in M. tuberculosis isolates susceptible to first-line drugs. RESULTS: There were 279 M. tuberculosis isolates from the two cohorts (2004-2005: 238 isolates; 2017: 41 isolates) that underwent OFX drug-susceptibility testing (critical concentration: 2 µg/ml). Of 238 isolates in Cohort 1, no resistance to OFX was detected (95% CI 0-0.016); likewise, in Cohort 2, no resistance to OFX was detected in 41 isolates (95% CI 0-0.086). Our findings suggest that FQ use remains a viable option for the treatment of first-line drug-susceptible TB in Peru.


Assuntos
Mycobacterium tuberculosis , Preparações Farmacêuticas , Tuberculose Resistente a Múltiplos Medicamentos , Tuberculose , Antituberculosos/farmacologia , Farmacorresistência Bacteriana , Fluoroquinolonas/farmacologia , Humanos , Levofloxacino , Testes de Sensibilidade Microbiana , Moxifloxacina , Peru/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia
7.
BMC Bioinformatics ; 22(1): 558, 2021 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-34798803

RESUMO

BACKGROUND: The ability to rapidly adapt to adverse environmental conditions represents the key of success of many pathogens and, in particular, of Mycobacterium tuberculosis. Upon exposition to heat shock, antibiotics or other sources of stress, appropriate responses in terms of genes transcription and proteins activity are activated leading part of a genetically identical bacterial population to express a different phenotype, namely to develop persistence. When the stress response network is mathematically described by an ordinary differential equations model, development of persistence in the bacterial population is associated with bistability of the model, since different emerging phenotypes are represented by different stable steady states. RESULTS: In this work, we develop a mathematical model of SigE stress response network that incorporates interactions not considered in mathematical models currently available in the literature. We provide, through involved analytical computations, accurate approximations of the system's nullclines, and exploit the obtained expressions to determine, in a reliable though computationally efficient way, the number of equilibrium points of the system. CONCLUSIONS: Theoretical analysis and perturbation experiments point out the crucial role played by the degradation pathway involving RseA, the anti-sigma factor of SigE, for coexistence of two stable equilibria and the emergence of bistability. Our results also indicate that a fine control on RseA concentration is a necessary requirement in order for the system to exhibit bistability.


Assuntos
Proteínas de Bactérias , Mycobacterium tuberculosis , Resposta ao Choque Térmico , Modelos Teóricos , Mycobacterium tuberculosis/genética , Fator sigma
8.
BMC Genomics ; 22(1): 841, 2021 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-34798821

RESUMO

BACKGROUND: Mycobacterium tuberculosis, the etiological agent of tuberculosis, has at least four ATP-Binding Cassette (ABC) transporters dedicated to carbohydrate uptake: LpqY/SugABC, UspABC, Rv2038c-41c, and UgpAEBC. LpqY/SugABC transporter is essential for M. tuberculosis survival in vivo and potentially involved in the recycling of cell wall components. The three-dimensional structures of substrate-binding proteins (SBPs) LpqY, UspC, and UgpB were described, however, questions about how these proteins interact with the cognate transporter are still being explored. Components of these transporters, such as SBPs, show high immunogenicity and could be used for the development of diagnostic and therapeutic tools. In this work, we used a phylogenetic and structural bioinformatics approach to compare the four systems, in an attempt to predict functionally important regions. RESULTS: Through the analysis of the putative orthologs of the carbohydrate ABC importers in species of Mycobacterium genus it was shown that Rv2038c-41c and UgpAEBC systems are restricted to pathogenic species. We showed that the components of the four ABC importers are phylogenetically separated into four groups defined by structural differences in regions that modulate the functional activity or the interaction with domain partners. The regulatory region in nucleotide-binding domains, the periplasmic interface in transmembrane domains and the ligand-binding pocket of the substrate-binding proteins define their substrates and segregation in different branches. The interface between transmembrane domains and nucleotide-binding domains show conservation of residues and charge. CONCLUSIONS: The presence of four ABC transporters in M. tuberculosis dedicated to uptake and transport of different carbohydrate sources, and the exclusivity of at least two of them being present only in pathogenic species of Mycobacterium genus, highlights their relevance in virulence and pathogenesis. The significant differences in the SBPs, not present in eukaryotes, and in the regulatory region of NBDs can be explored for the development of inhibitory drugs targeting the bacillus. The possible promiscuity of NBDs also contributes to a less specific and more comprehensive control approach.


Assuntos
Mycobacterium tuberculosis , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carboidratos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Filogenia
9.
BMC Genomics ; 22(1): 844, 2021 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-34802420

RESUMO

BACKGROUND: Tuberculosis is one of the deadliest disease caused by Mycobacterium tuberculosis. Its treatment still becomes a burden for many countries including Indonesia. Drug resistance is one of the problems in TB treatment. However, a development in the molecular field through Whole-genome sequencing (WGS) can be used as a solution in detecting mutations associated with TB- drugs. This investigation intended to implement this data for supporting the scientific community in deeply understanding any TB epidemiology and evolution in Papua along with detecting any mutations in genes associated with TB-Drugs. RESULT: A whole-genome sequencing was performed on the random samples from TB Referral Laboratory in Papua utilizing MiSeq 600 cycle Reagent Kit (V3). Furthermore, TBProfiler was used for genome analysis, RAST Server was employed for annotation, while Gview server was applied for BLAST genome mapping and a Microscope server was implemented for Regions of Genomic Plasticity (RGP). The largest genome of M. tuberculosis obtained was at the size of 4,396,040 bp with subsystems number at 309 and the number of coding sequences at 4326. One sample (TB751) contained one RGP. The drug resistance analysis revealed that several mutations associated with TB-drug resistance existed. In details, mutations of rpoB gene which were identified as S450L, D435Y, H445Y, L430P, and Q432K had caused the reduced effectiveness of rifampicin; while the mutases in katG (S315T), kasA (312S), inhA (I21V), and Rv1482c-fabG1 (C-15 T) genes had contributed to the resistance in isoniazid. In streptomycin, the resistance was triggered by the mutations in rpsL (K43R) and rrs (A514C, A514T) genes, and, in Amikacin, its resistance was led by mutations in rrs (A514C) gene. Additionally, in Ethambutol and Pyrazinamide, their reduced effectiveness was provoked by embB gene mutases (M306L, M306V, D1024N) and pncA (W119R). CONCLUSIONS: The results from whole-genome sequencing of TB clinical sample in Papua, Indonesia could contribute to the surveillance of TB-drug resistance. In the drug resistance profile, there were 15 Multi Drugs Resistance (MDR) samples. However, Extensively Drug-resistant (XDR) samples have not been found, but samples were resistant to only Amikacin, a second-line drug.


Assuntos
Mycobacterium tuberculosis , Preparações Farmacêuticas , Tuberculose Resistente a Múltiplos Medicamentos , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla , Humanos , Indonésia , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/genética
10.
Int J Nanomedicine ; 16: 7339-7352, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34754188

RESUMO

Background: Mycobacterium tuberculosis' rapid detection is still a formidable challenge to have control over the lethal disease. New diagnostic methods such as LED fluorescence microscopy, Genexpert, Interferon Gamma Release Assay (IGRA) are limited on efficacy spectrum owing to their high cost, time-intensive and laborious nature, in addition their low sensitivity hinders their robustness and portability. Electroanalytical methods are now being considered as an excellent alternative, being currently employed for efficient detection of the analytes with the potential of being portable. This report suggests label-free electrochemical detection of Mycobacterium tuberculosis (Mtb) via its marker, insertion sequence (IS6110). Methods: In this pursuit, graphene oxide-chitosan nanocomposite (GO-CHI), a biocompatible matrix, having a large electroactive area with an overall positively charged surface, is fabricated and characterized. The obtained GO-CHI nanocomposite is then immobilized on the ITO surface to form a positively functionalized electrochemical sensor for the detection of Mtb. DNA probe, specific for the IS6110, was electrostatically anchored on a positively charged electrode surface and the resistance of charge transfer was investigated for the sensitive and specific (complementary vs non-complementary) detection of Mtb by cyclic voltammetry and differential pulse voltammetry techniques. Results: The cyclic voltammetry was found to be diffusion controlled facilitating the absorption of analyte on the electrode surface. The label-free "genosensor" was found to detect a hybridization efficiency with a limit of detection of 3.4 pM, and correlation coefficient R2=0.99 when analysed over a range of concentrations of DNA from 7.86 pM to 94.3pM. The genosensor was also able to detect target DNA from raw sputum samples of clinical isolates without DNA purification. Conclusion: This electrochemical genosensor provides high sensitivity and specificity; thus offering a promising platform for clinical diagnosis of TB and other infectious diseases in general.


Assuntos
Técnicas Biossensoriais , Grafite , Mycobacterium tuberculosis , Nanocompostos , Técnicas Eletroquímicas , Limite de Detecção , Mycobacterium tuberculosis/genética
11.
PLoS One ; 16(11): e0258743, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34758029

RESUMO

BCG vaccination is known to induce innate immune memory, which confers protection against heterologous infections. However, the effect of BCG vaccination on the conventional adaptive immune cells subsets is not well characterized. We investigated the impact of BCG vaccination on the frequencies of T cell subsets and common gamma c (γc) cytokines in a group of healthy elderly individuals (age 60-80 years) at one month post vaccination as part of our clinical study to examine the effect of BCG on COVID-19. Our results demonstrate that BCG vaccination induced enhanced frequencies of central (p<0.0001) and effector memory (p<0.0001) CD4+ T cells and diminished frequencies of naïve (p<0.0001), transitional memory (p<0.0001), stem cell memory (p = 0.0001) CD4+ T cells and regulatory T cells. In addition, BCG vaccination induced enhanced frequencies of central (p = 0.0008), effector (p<0.0001) and terminal effector memory (p<0.0001) CD8+ T cells and diminished frequencies of naïve (p<0.0001), transitional memory (p<0.0001) and stem cell memory (p = 0.0034) CD8+T cells. BCG vaccination also induced enhanced plasma levels of IL-7 (p<0.0001) and IL-15 (p = 0.0020) but diminished levels of IL-2 (p = 0.0033) and IL-21 (p = 0.0020). Thus, BCG vaccination was associated with enhanced memory T cell subsets as well as memory enhancing γc cytokines in elderly individuals, suggesting its ability to induce non-specific adaptive immune responses.


Assuntos
Vacina BCG/imunologia , Citocinas/imunologia , Memória Imunológica/imunologia , Subunidade gama Comum de Receptores de Interleucina/imunologia , Idoso , Idoso de 80 Anos ou mais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , Feminino , Humanos , Interleucinas/imunologia , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Vacinação/métodos
12.
Front Cell Infect Microbiol ; 11: 707244, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34778100

RESUMO

The GeneLEAD VIII (Diagenode, Belgium) is a new, fully automated, sample-to-result precision instrument for the extraction of DNA and PCR detection of Mycobacterium tuberculosis complex (MTBC) directly from clinical samples. The Deeplex Myc-TB® assay (Genoscreen, France) is a diagnostic kit based on the deep sequencing of a 24-plexed amplicon mix allowing simultaneously the detection of resistance to 13 antituberculous (antiTB) drugs and the determination of spoligotype. We evaluated the performance of a strategy combining the both mentioned tools to detect directly from clinical samples, in 8 days, MTBC and its resistance to 13 antiTB drugs, and identify potential transmission of strains from patient-to-patient. Using this approach, we screened 112 clinical samples (65 smear-negative) and 94 MTBC cultured strains. The sensitivity and the specificity of the GeneLEAD/Deeplex Myc-TB approach for MTBC detection were 79.3% and 100%, respectively. One hundred forty successful Deeplex Myc-TB results were obtained for 46 clinical samples and 94 strains, a total of 85.4% of which had a Deeplex Myc-TB susceptibility and resistance prediction consistent with phenotypic drug susceptibility testing (DST). Importantly, the Deeplex Myc-TB assay was able to detect 100% of the multidrug-resistant (MDR) MTBC tested. The lowest concordance rates were for pyrazinamide, ethambutol, streptomycin, and ethionamide (84.5%, 81.5%, 73%, and 55%, respectively) for which the determination of susceptibility or resistance is generally difficult with current tools. One of the main difficulties of Deeplex Myc-TB is to interpret the non-synonymous uncharacterized variants that can represent up to 30% of the detected single nucleotide variants. We observed a good level of concordance between Deeplex Myc-TB-spoligotyping and MIRU-VNTR despite a lower discriminatory power for spoligotyping. The median time to obtain complete results from clinical samples was 8 days (IQR 7-13) provided a high-throughput NGS sequencing platform was available. Our results highlight that the GeneLEAD/Deeplex Myc-TB approach could be a breakthrough in rapid diagnosis of MDR TB in routine practice.


Assuntos
Mycobacterium tuberculosis , Preparações Farmacêuticas , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , DNA , Resistência a Medicamentos , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/genética
14.
Indian J Med Res ; 154(1): 85-89, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34782533

RESUMO

Background & objectives: Linezolid (LZD) is increasingly being used in tuberculosis (TB) treatment. However, LZD resistance has already been reported, which is highly alarming, given its critical therapeutic role. This study was aimed to phenotypically and genotypically assess LZD resistance in Mycobacterium tuberculosis (MTB) isolates at a laboratory in a tertiary care centre in Mumbai, India. Methods: A sample of 32 consecutive LZD-resistant MTB isolates identified by liquid culture susceptibility testing was subjected to whole-genome sequencing (WGS) on the Illumina NextSeq platform. Sequences were analyzed using BioNumerics software to predict resistance for 12 antibiotics within 15 min. Results: Sixty eight of the 2179 isolates tested for LZD resistance by MGIT-based susceptibility testing (June 2015 to June 2016) were LZD-resistant. Thirty two consecutive LZD-resistant isolates were analyzed by WGS to screen for known mutations conferring LZD resistance. WGS of 32 phenotypically LZD-resistant isolates showed that C154R in the rplC gene and G2814T in the rrl gene were the major resistance determinants. Interpretation & conclusions: LZD resistance poses an important risk to the success of treatment regimens, especially those designed for resistant isolates; such regimens are extensively used in India. As LZD-containing regimens increase in prominence, it is important to support clinical decision-making with an improved understanding of the common mutations conferring LZD resistance and their frequency in different settings.


Assuntos
Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Farmacorresistência Bacteriana Múltipla , Humanos , Linezolida/farmacologia , Linezolida/uso terapêutico , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/genética , Centros de Atenção Terciária , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/genética
16.
Front Cell Infect Microbiol ; 11: 681131, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34790584

RESUMO

Co-infection of Mycobacterium tuberculosis and Paracoccidioides brasiliensis, present in 20% in Latin America, is a public health problem due to a lack of adequate diagnosis. These microorganisms are capable of forming biofilms, mainly in immunocompromised patients, which can lead to death due to the lack of effective treatment for both diseases. The present research aims to show for the first time the formation of mixed biofilms of M. tuberculosis and P. brasiliensis (Pb18) in vitro, as well as to evaluate the action of 3'hydroxychalcone (3'chalc) -loaded nanoemulsion (NE) (NE3'chalc) against monospecies and mixed biofilms, the formation of mixed biofilms of M. tuberculosis H37Rv (ATCC 27294), 40Rv (clinical strains) and P. brasiliensis (Pb18) (ATCC 32069), and the first condition of formation (H37Rv +Pb18) and (40Rv + Pb18) and second condition of formation (Pb18 + H37Rv) with 45 days of total formation time under both conditions. The results of mixed biofilms (H37Rv + Pb18) and (40Rv + Pb18), showed an organized network of M. tuberculosis bacilli in which P. brasiliensis yeasts are connected with a highly extracellular polysaccharide matrix. The (Pb18 + H37Rv) showed a dense biofilm with an apparent predominance of P. brasiliensis and fragments of M. tuberculosis. PCR assays confirmed the presence of the microorganisms involved in this formation. The characterization of NE and NE3'chalc displayed sizes from 145.00 ± 1.05 and 151.25 ± 0.60, a polydispersity index (PDI) from 0.20± 0.01 to 0.16± 0.01, and zeta potential -58.20 ± 0.92 mV and -56.10 ± 0.71 mV, respectively. The atomic force microscopy (AFM) results showed lamellar structures characteristic of NE. The minimum inhibitory concentration (MIC) values of 3'hidroxychalcone (3'chalc) range from 0.97- 7.8 µg/mL and NE3'chalc from 0.24 - 3.9 µg/mL improved the antibacterial activity when compared with 3'chalc-free, no cytotoxicity. Antibiofilm assays proved the efficacy of 3'chalc-free incorporation in NE. These findings contribute to a greater understanding of the formation of M. tuberculosis and P. brasiliensis in the mixed biofilm. In addition, the findings present a new possible NE3'chalc treatment alternative for the mixed biofilms of these microorganisms, with a high degree of relevance due to the lack of other treatments for these comorbidities.


Assuntos
Mycobacterium tuberculosis , Paracoccidioides , Biofilmes , Humanos , Testes de Sensibilidade Microbiana
17.
BMJ Case Rep ; 14(11)2021 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-34728509

RESUMO

A 33-year-old man presented with a 2-year history of right knee swelling with fungating masses and white-yellow discharge. Severe pain, limited movement and signs of sepsis were absent. Debridement, partial synovectomy and arthrotomy were done for the multiple sinuses that developed over the knee. Synovial tissue analysis yielded a positive acid-fast bacillus smear and Mycobacterium tuberculosis PCR test, while aerobic culture studies grew Pseudomonas aeruginosa and Acinetobacter baumannii Chronic granulomatous inflammation was seen on histopathology. Alongside antibiotic therapy, multiple debridements of the right knee were required to eradicate the infection and allow wound repair. A flap coverage with split-thickness skin graft was performed after the bacterial infection resolved, and the patient was discharged ambulatory with minimal pain. Such atypical presentations of monarthritis require immediate workup and a prompt referral to a multidisciplinary team to establish the diagnosis and initiate appropriate management before irreversible joint destruction and disability ensues.


Assuntos
Artrite Infecciosa , Mycobacterium tuberculosis , Tuberculose Osteoarticular , Adulto , Antibacterianos/uso terapêutico , Artrite Infecciosa/diagnóstico , Artrite Infecciosa/tratamento farmacológico , Humanos , Articulação do Joelho/diagnóstico por imagem , Articulação do Joelho/cirurgia , Masculino , Tuberculose Osteoarticular/diagnóstico , Tuberculose Osteoarticular/tratamento farmacológico
18.
Front Immunol ; 12: 656419, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34745081

RESUMO

Tuberculosis (TB) is the global health problem with the second highest number of deaths from a communicable disease after COVID-19. Although TB is curable, poor health infrastructure, long and grueling TB treatments have led to the spread of TB pandemic with alarmingly increasing multidrug-resistant (MDR)-TB prevalence. Alternative host modulating therapies can be employed to improve TB drug efficacies or dampen the exaggerated inflammatory responses to improve lung function. Here, we investigated the adjunct therapy of natural immune-modulatory compound berberine in C57BL/6 mouse model of pulmonary TB. Berberine treatment did not affect Mtb growth in axenic cultures; however, it showed increased bacterial killing in primary murine bone marrow-derived macrophages and human monocyte-derived macrophages. Ad libitum berberine administration was beneficial to the host in combination with rifampicin and isoniazid. Berberine adjunctive treatment resulted in decreased lung pathology with no additive or synergistic effects on bacterial burdens in mice. Lung immune cell flow cytometry analysis showed that adjunctive berberine treatment decreased neutrophil, CD11b+ dendritic cell and recruited interstitial macrophage numbers. Late onset of adjunctive berberine treatment resulted in a similar phenotype with consistently reduced numbers of neutrophils both in lungs and the spleen. Together, our results suggest that berberine can be supplemented as an immunomodulatory agent depending on the disease stage and inflammatory status of the host.


Assuntos
Antituberculosos/uso terapêutico , Berberina/uso terapêutico , Fatores Imunológicos/uso terapêutico , Isoniazida/uso terapêutico , Rifampina/uso terapêutico , Tuberculose Pulmonar/tratamento farmacológico , Animais , Antituberculosos/farmacologia , Berberina/farmacologia , Citocinas/imunologia , Células Dendríticas/efeitos dos fármacos , Quimioterapia Combinada , Feminino , Humanos , Fatores Imunológicos/farmacologia , Isoniazida/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Rifampina/farmacologia , Baço/efeitos dos fármacos , Baço/imunologia , Baço/microbiologia , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologia
19.
Anal Chim Acta ; 1186: 339090, 2021 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-34756272

RESUMO

Herein, a universal fluorescent biosensor was developed for detecting Mycobacterium Tuberculosis (MTB) specific insertion sequence IS6110 gene fragment based on Förster resonance energy transfer (FRET) strategy. CdTe quantum dots (QDs), with excellent luminous performance, were used to label single-stranded DNA (ssDNA) as fluorescence donor (QDs-DNA), in which the ssDNA was complementary to the IS6110 gene fragment. A new type of two-dimensional metal-organic framework (Cu-TCPP) was served as an acceptor. The Cu-TCPP exhibited a higher affinity towards ssDNA than double-stranded DNA (dsDNA). In the absence of targets, the fluorescence of QDs-DNA was quenched - due to the π-π stacking interactions between Cu-TCPP and ssDNA. Otherwise, QDs-DNA hybridized with the target to form a double helix and the fluorescence maintained in a target-concentration dependent manner. Excess QDs-DNA would be quenched and produced negligible background signal. The fluorescent sensor possessed a linear range from 0.05 nM to 1.0 nM with a low detection limit of 35 pM. Furthermore, we successfully applied this biosensing system to detect clinical sputum samples. This method displayed high sensitivity, specificity and great potentials in the early diagnosis of Tuberculosis.


Assuntos
Técnicas Biossensoriais , Compostos de Cádmio , Estruturas Metalorgânicas , Mycobacterium tuberculosis , Pontos Quânticos , Sondas de DNA , Transferência Ressonante de Energia de Fluorescência , Mycobacterium tuberculosis/genética , Telúrio
20.
J Med Invest ; 68(3.4): 220-227, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34759134

RESUMO

The incidence rate of pulmonary nontuberculous mycobacterial disease (PNTMD) in Japan is the highest among major industrialized nations. Although the typical clinical course and radiological manifestations of PNTMD are different from those of pulmonary tuberculosis (TB), confusion about these mycobacterial diseases leads to a diagnostic pitfall. Diagnostic challenges include the coexistence of Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM), false positives for NTM in MTB nucleic acid amplification tests, microbial substitution, and abnormal radiological manifestations caused by NTM. Features of extrapulmonary NTM diseases, such as pleurisy, vertebral osteomyelitis, and disseminated disease, are different from the corresponding tuberculous diseases. Moreover, the immunological background of the patient (status of human immunodeficiency virus infection with or without antiviral therapy, continuation or discontinuation of immunosuppressive therapy, use of immune checkpoint inhibitor, pregnancy and delivery, etc.) influences the pathophysiology of mycobacterial diseases. This review describes the varying clinical presentations of NTM disease with emphasis on the differences from TB. J. Med. Invest. 68 : 220-227, August, 2021.


Assuntos
Infecções por Mycobacterium não Tuberculosas , Mycobacterium tuberculosis , Tuberculose Pulmonar , Tuberculose , Humanos , Infecções por Mycobacterium não Tuberculosas/diagnóstico por imagem , Micobactérias não Tuberculosas , Tuberculose Pulmonar/diagnóstico por imagem
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