An ultra-sensitive "turn-off" fluorescent sensor for the trace detection of rifampicin based on glutathione-stabilized copper nanoclusters.

Rifampicin is a common antibiotic used in human and veterinary medicine to treat tuberculosis and other diseases caused by numerous pathogenic bacteria. However, the excessive or improper use of rifampicin usually leads to a series of problems, including bacterial resistance, excessive drug-resistance and water pollution. Thus, it is of great importance to develop selective and sensitive assays for monitoring rifampicin in biological systems. In this study, we designed a fluorescence "turn-off" strategy for the trace detection of rifampicin based on a glutathione-stabilized copper nanoclusters (GSH-Cu NC) sensor. In an aqueous solution, the fluorescence of the GSH-Cu NCs at 632 nm can be quenched effectively and selectively by rifampicin due to the inner-filter effect (IFE) of fluorescence mechanism. Distinctively, this GSH-Cu NC sensor exhibited excellent fluorescence sensing capability for the trace detection of rifampicin with a very low limit of detection (LOD) of 16 pM in a wide linear range from 50 to 10 000 pM. It is not only more sensitive than the other methods previously reported for the detection of rifampicin, but also has an outstanding selectivity and strong anti-interference in complex samples. Furthermore, the as-developed GSH-Cu NCs were also successfully applied to determine rifampicin in different real samples with quantitative spike recoveries ranging from 97% to 105%.

Fluorescence enhancement of glutathione capped CdTe/ZnS quantum dots by embedding into cationic starch for sensitive detection of rifampicin.

In this study, we describe the synthesis of a new quantum dots (QDs) by embedding glutathione capped CdTe/ZnS QDs into cationic starch biopolymer (CS-GSH-CdTe/ZnS QDs). The fluorescence intensity of prepared QDs was significantly enhanced. When QDs interacted with rifampicin, the fluorescence intensity of the CS-GSH-CdTe/ZnS QDs was highly quenched compared with GSH-CdTe/ZnS QDs. Based on the above, a new fluorescent nanosensor for simple, sensitive and selective detection of rifampicin was developed. The fluorescence quenching was well described by the typical Stern-Volmer equation. After optimization, the linear range of the as-prepared QDs fluorescence intensity versus the concentration of rifampicin was F0/F=0.0422Q+1.109 (R2=0.99). The detection limit was 0.06×10-6mol/L. The proposed method with satisfactory results was used to detect rifampicin in commercial capsules and tablets.

Novel electrochemical biosensor based on PVP capped CoFe2O4@CdSe core-shell nanoparticles modified electrode for ultra-trace level determination of rifampicin by square wave adsorptive stripping voltammetry.

This work introduces a new electrochemical sensor based on polyvinyl pyrrolidone capped CoFe2O4@CdSe core-shell modified electrode for a rapid detection and highly sensitive determination of rifampicin (RIF) by square wave adsorptive stripping voltammetry. The new PVP capped CoFe2O4@CdSe with core-shell nanostructure was synthesized by a facile synthesis method for the first time. PVP can act as a capping and etching agent for protection of the outer surface nanoparticles and formation of a mesoporous shell, respectively. Another important feature of this work is the choice of the ligand (1,10-phenanthroline) for precursor cadmium complex that works as a chelating agent in order to increase optical and electrical properties and stability of prepared nanomaterial. The nanoparticles have been characterized by field emission scanning electron microscopy (FESEM), transmission electron microscope (TEM), energy dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), UV-vis, photoluminescence (PL) spectroscopy, FT-IR, and cyclic voltammetry techniques. The PL spectroscopy study of CoFe2O4@CdSe has shown significant PL quenching by the formation of CoFe2O4 core inside CdSe, this shows that CoFe2O4 NPs are efficient electron acceptors with the CdSe. It is clearly observed that the biosensor can significantly enhance electrocatalytic activity towards the oxidation of RIF, under the optimal conditions. The novelty of this work arises from the new synthesis method for the core-shell of CoFe2O4@CdSe. Then, the novel electrochemical biosensor was fabricated for ultra-trace level determination of rifampicin with very low detection limit (4.55×10-17M) and a wide linear range from 1.0×10-16 to 1.0×10-7M. The fabricated biosensor showed high sensitivity and selectivity, good reproducibility and stability. Therefore, it was successfully applied for the determination of ultra-trace RIF amounts in biological and pharmaceutical samples with satisfactory recovery data.

Nickel hydroxide nanoparticles-reduced graphene oxide nanosheets film: layer-by-layer electrochemical preparation, characterization and rifampicin sensory application.

Electrochemical deposition, as a well-controlled synthesis procedure, has been used for subsequently layer-by-layer preparation of nickel hydroxide nanoparticle-reduced graphene oxide nanosheets (Ni(OH)2-RGO) on a graphene oxide (GO) film pre-cast on a glassy carbon electrode surface. The surface morphology and nature of the nano-hybrid film (Ni(OH)2-RGO) was thoroughly characterized by scanning electron and atomic force microscopy, spectroscopy and electrochemical techniques. The modified electrode appeared as an effective electro-catalytic model for analysis of rifampicin (RIF) by using linear sweep voltammetry (LSV). The prepared modified electrode exhibited a distinctly higher activity for electro-oxidation of RIF than either GO, RGO nanosheets or Ni(OH)2 nanoparticles. Enhancement of peak currents is ascribed to the fast heterogeneous electron transfer kinetics that arise from the synergistic coupling between the excellent properties of RGO nanosheets (such as high density of edge plane sites, subtle electronic characteristics and attractive π-π interaction) and unique properties of metal nanoparticles. Under the optimized analysis conditions, the modified electrode showed two oxidation processes for rifampicin at potentials about 0.08 V (peak I) and 0.69 V (peak II) in buffer solution of pH 7.0 with a wide linear dynamic range of 0.006-10.0 µmol L(-1) and 0.04-10 µmol L(-1) with a detection limit of 4.16 nmol L(-1) and 2.34 nmol L(-1) considering peaks I and II as an analytical signal, respectively. The results proved the efficacy of the fabricated modified electrode for simple, low cost and highly sensitive medicine sensor well suited for the accurate determinations of trace amounts of rifampicin in the pharmaceutical and clinical preparations.

Penetration of anti-infective agents into pulmonary epithelial lining fluid: focus on antifungal, antitubercular and miscellaneous anti-infective agents.

Epithelial lining fluid (ELF) is often considered to be the site of extracellular pulmonary infections. During the past 25 years, a limited number of studies have evaluated the intrapulmonary penetration of antifungal, antitubercular, antiparasitic and antiviral agents. For antifungal agents, differences in drug concentrations in ELF or bronchoalveolar lavage (BAL) fluid were observed among various formulations or routes of administration, and between agents within the same class. Aerosolized doses of deoxycholate amphotericin B, liposomal amphotericin B and amphotericin B lipid complex resulted in higher concentrations in ELF or BAL fluid than after intravenous administration. The mean concentrations in ELF following intravenous administration of both anidulafungin and micafungin ranged between 0.04 and 1.38 µg/mL, and the ELF to plasma concentration ratios (based on the area under the concentration-time curve for total drug concentrations) were between 0.18 and 0.22 during the first 3 days of therapy. Among the azole agents, intravenous administration of voriconazole resulted in the highest mean ELF concentrations (range 10.1-48.3 µg/mL) and ratio of penetration (7.1). The range of mean ELF concentrations of itraconazole and posaconazole following oral administration was 0.2-1.9 µg/mL, and the ELF to plasma concentration ratios were <1. A series of studies have evaluated the intrapulmonary penetration of first- and second-line oral antitubercular agents in healthy adult subjects and patients with AIDS. The ELF to plasma concentration ratio was >1 for isoniazid, ethambutol, pyrazinamide and ethionamide. For rifampicin (rifampin) and rifapentine, the ELF to plasma concentration ratio ranged between 0.2 and 0.32, but in alveolar macrophages the concentration of rifampicin was much higher (145-738 µg/mL compared with 3.3-7.5 µg/mL in ELF). No intrapulmonary studies have been conducted for rifabutin. Sex, AIDS status or smoking history had no significant effects on the magnitude of ELF concentrations of antitubercular agents. Subjects who were slow acetylators had higher plasma and ELF concentrations of isoniazid than those who were fast acetylators. Penetration of dapsone into ELF was very good, with the range of mean ELF to plasma concentration ratios being 0.65-2.91 at individual sampling times over 48 hours. Once-daily dosing of aerosolized pentamidine resulted in higher concentrations in BAL fluid than after intravenous administration. The mean BAL concentrations at 15-32 days after once- or twice-monthly administration of aerosolized pentamidine 300 and 600 mg ranged from 6.5 to 28.4 ng/mL. No differences in pentamidine BAL concentrations were observed in symptomatic patients who developed Pneumocystis jirovecii pneumonia compared with patients who did not. Zanamivir concentrations in ELF were similar in magnitude (range 141-326 ng/mL) following administration by continuous intravenous infusion (3 mg/hour), oral inhalation (10 mg every 12 hours) and intravenous bolus (200 mg every 12 hours). Data from case reports have suggested that concentrations of nelfinavir and saquinavir in ELF are undetectable, whereas tipranavir and lopinavir had measureable ELF concentrations (2.20 µmol/L and 14.4 µg/mL, respectively) when these protease inhibitors were co-administrated with ritonavir. While the clinical significance of ELF or BAL concentrations remains unknown for this group of anti-infective agents, the knowledge of drug penetration into the extracellular space of the lung should assist in re-evaluating and designing specific dosing regimens for use against potential pathogens.

Development and validation of an electroanalytical methodology for determination of isoniazid and rifampicin content in pharmaceutical formulations / Desenvolvimento e validação de uma metodologia eletroanalítica para a determinação do teor de isoniazida e rifampicina em preparações farmacêuticas

Tuberculosis remains a major public health problem, especially in developing countries. Brazil presents the largest number of cases in Latin America and is among the 22 countries considered priorities by the World Health Organization (WHO). The Rio de Janeiro state has the largest number of cases registered in the country. The treatment of patients, commonly, makes use of the drugs isoniazid and rifampicin for six months. This study aimed to develop and validate an electroanalytical methodology, using the technique of differential pulse voltammetry for the determination of these drugs in the associated form, in order to evaluate the quality of medicines distributed in the state of Rio de Janeiro. The potential reduction for the isoniazid and rifampicin were -1.10 and -0.90 V. The developed and validated electroanalytical method presented a linear range of 0.25 to 1.25 mg/L to isoniazid, limits of detection and quantification of 0.05 and 0.14 mg/L, and recovery of 98.2 ± 0.4 percent; a tracking linear of 0.40 to 2.00 mg/L for rifampicin, with limits of detection and quantification of 0.07 and 0.19 mg/L and recovery of 95.8 ± 0.6 percent. Six lots of medicines from two pharmaceutical companies were analyzed. Only one of the samples showed unsatisfactory levels of rifampicin.

A tuberculose continua sendo um importante problema de saúde pública, especialmente em países em desenvolvimento. O Brasil apresenta o maior número de casos da América Latina, estando entre os 22 países considerados prioritários nas ações de controle da doença pela Organização Mundial da Saúde (OMS). No Brasil, o Rio de Janeiro é o estado com o maior número de casos registrados no país. O tratamento de doentes com tuberculose faz uso dos fármacos isoniazida e rifampicina durante seis meses. O presente trabalho objetivou desenvolver e validar metodologia eletroanalítica, utilizando a técnica de voltametria de pulso diferencial, para a determinação desses dois princípios ativos na forma associada e avaliar a qualidade dos medicamentos distribuídos no estado do Rio de Janeiro. Os potenciais de redução para a isoniazida e rifampicina foram respectivamente -1,10 e -0,90 V. O método eletroanalítico desenvolvido e validado apresentou para a isoniazida faixa linear de 0,25 a 1,25 mg/L, limites de detecção e quantificação de 0,05 e 0,14 mg/L e recuperação de 98,2 ± 0,4 por cento; para a rifampicina faixa linear de 0,40 a 2,00 mg/L, limites de detecção e quantificação de 0,07 e 0,19 mg/L e recuperação de 95,8 ± 0,6 por cento. Foram analisados 6 lotes de medicamentos de dois laboratórios farmacêuticos. Apenas uma das amostras apresentou teor de rifampicina insatisfatório.

Mutações no gene nat de isolados de Mycobacterium tuberculosis: efeito na atividade enzimática e no perfil de resistência à isoniazida / Mutation in the nat gene of Mycobacterium tuberculosis strains: effect on the enzymatic activity and on the isoniazid-resistance profile

Como entre 25%-50% dos isolados de Mycobacterium tuberculosis resistentes à INH não apresentam mutações nos genes katG, inhA, ahpC e kasA que possam justificar sua resistência, foi proposta a influência de mutações específicas no gene nat nos mecanismos de resistência e atividade da NAT. Todos os isolados obtidos (n=125) foram identificados e caracterizados através da amplificação pela PCR da IS6110 e por MIRU-VNTR, respectivamente. A determinação da concentração inibitória mínima (CIM) foi realizada pelo método REMA. Após triagem de mutações nos genes caracteristicamente envolvidos com resistência pela PCR-SSCP, seguida de seqüenciamento de DNA, foram selecionados 45 isolados para o estudo de mutações específicas (pela PCR e sequenciamento) e expressão gênica do mRNA do gene nat através da RT-PCR em tempo real. Confirmou-se que mutação no gene katG é a mais correlacionada com a resistência à INH, pois 68,4% das cepas resistentes apresentaram mutação neste gene. Mutações na região promotora do gene inhA, na região intergênica oxyR-ahpC e no gene kasA foram encontradas em 8,8%, 5,6% e 21,6% dos isolados, respectivamente...

Detección de mutaciones en los Genes rpoß y katG asociados a resistencia en cepas de Mycobacterium tuberculosis de Bolivia / Detection of mutations in rpoß and katG genes associated to resistance in Mycobacterium tuberculosis strains from Bolivia

Objetivo: Evaluar la sensibilidad y especificidad de la genotipificación, como instrumento de diagnóstico rápido y confiable parala detección de mutaciones en los genes rpoß y katG asociados a resistencia,en cepas de Mycobacterium tuberculosis de Bolivia.Diseño: Test Diagnóstico Metodología: Las cepas analizadas fueron aisladas y enviadas por los diferentes Laboratorios de la Red Nacional de Diagnósticode Tuberculosis de Bolivia entre febrero y diciembre de 2007. La muestra para el presente estudio estuvo constituida por un totalde 65 aislamientos previamente caracterizados por métodos fenotípicos de cultivo y pruebas de sensibilidad a la RIF e INH, porel método de las proporciones Canetti-Rist. La genotipificación ha sido realizada utilizando el kit Genotype MTBDR, basado enla utilización de métodos de amplificación e hibridización, para detectar mutaciones a nivel de los marcadores de resistencia rpoßy katG.Resultados: Se procedió al cálculo de la sensibilidad y especificidad de la prueba de diagnóstico; además de los valores predictivospositivo y negativo. Dicho análisis muestra los siguientes resultados: sensibilidad 74...

Objective:To evaluate the sensitivity and specificity of genotyping as a tool for rapid and reliable detection of mutations in rpoß and katG genes associated with resistance in Mycobacterium tuberculosisstrains from Bolivia. Design:Diagnostic Test Methodology: The strains analyzed were isolated and submitted by different laboratories of the National Network for Diagnosisof Tuberculosis of Bolivia between February and December 2007. The sample for this study consisted of 65 isolates previousl y characterized by phenotypic methods of culture and sensitivity testing to RIF and INH by the Canetti-Rist proportion method. Genotyping of these samples has been done using the MTBDR Genotype kit, according to amplification and hybridization methodsto detect mutations at the rpoß and katG resistance markers.Results:The sensitivity and specificity of the diagnostic tests were calculated, as well as the positive and negative predictivevalues. This analysis shows the following results: sensitivity 74%, specificity 92%, positive predictive value 92%, and negative predictive value 73%. Conclusions: The genotyping test using Genotype MTBDR, meeting validation criteria for a diagnostic test study in our country,constitutes a quick, useful and reliable tool for use in diagnosis and routine determination of sensitivity and resistance in MTBCstrains.

Intracellular growth and drug susceptibility of Mycobacterium tuberculosis in macrophages.

Intracellular growth of Mycobacterium tuberculosis in human and rabbit monocytes and in mouse and guinea pig macrophages was evaluated. Monocytes or macrophages were infected with M. tuberculosis H37Rv with the multiplicity of infection at 10 mycobacteria per monocyte. The average percentages of infected human and rabbit monocytes were 22% and 19%, while mouse and guinea pig macrophages were 46% and 58%, respectively. The average generation times of M. tuberculosis H37Rv inside human and rabbit monocytes and in mouse and guinea pig macrophages, after culturing the infected cells for 10 days, were 33.4, 50.3, 31.4, and 25.6 h, respectively. Using infected guinea pig macrophages for intracellular evaluation of drug susceptibility, the minimal inhibitory concentrations (MICs) of isoniazid to the intracellular and extracellular M. tuberculosis H37Rv were 0.1 and 0.4 microg/ml, while the MICs of rifampicin were 0.1 and 0.2 microg/ml, respectively. The minimal bactericidal concentrations (MBCs) of isoniazid to the intracellular and extracellular H37Rv were 0.2 and 0.4 microg/ml, while the MCSs of rifampicin were 0.1 and 0.2 microg/ml, respectively.

HRP-based biosensor for monitoring rifampicin.

Pyrrole was electropolymerized onto a Pt electrode in the presence of LiClO(4) and horseradish peroxidase (HRP). This HRP-based biosensor has been used for the amperometric detection of rifampicin (RIF) in the presence of a constant concentration of H(2)O(2). The C(H(2)O(2)) as well as the applied potential (E(ap)) and the pH of the phosphate buffer have simultaneously been optimized through a central composite design. Under these conditions, repeatability, reproducibility, and stability of the modified electrode have been analyzed. The detection limit for RIF has been calculated taking into account the probability of false-positive (alpha) and -negative (beta), reaching a value of 5.06x10(-6) mol dm(-3). The biosensor was applied to the determination of RIF in pharmaceutical preparations and biological samples.

Rifampin concentrations in various compartments of the human brain: a novel method for determining drug levels in the cerebral extracellular space.

Antimicrobial therapy for brain infections is notoriously difficult because of the limited extent of knowledge about drug penetration into the brain. Therefore, we determined the penetration of rifampin into various compartments of the human brain, including the cerebral extracellular space (CES). Patients undergoing craniotomy for resection of primary brain tumors were given a standard dose of 600 mg of rifampin intravenously before the operation. A microdialysis probe (10 by 0.5 mm) was inserted into the cortex distantly from the resection and was perfused with two different rifampin solutions. Rifampin concentrations in the CES were calculated by the no-net-flux method. Intraoperatively, samples were taken from brain tumor tissue, perifocal tissue, and normal brain tissue in the case of pole resections. Rifampin concentrations in the various samples were determined by using a bioassay with Sarcinea lutea. In the various compartments, rifampin concentrations were highest within tumors (1.37 +/- 1.34 microg/ml; n = 8), followed by the perifocal region (0.62 +/- 0.67 microg/ml; n = 8), the CES (0.32 +/- 0.11 microg/ml; n = 6), and normal brain tissue (0.29 +/- 0.15 microg/ml; n = 7). Rifampin concentrations in brain tumors do not adequately reflect concentrations in normal brain tissue or in the CES. Rifampin concentrations in the CES, as determined by microdialysis, are the most reproducible, and the least scattered, of the values for all compartments evaluated. Rifampin concentrations in all compartments exceed the MIC for staphylococci and streptococci. However, CES concentrations may be below the MICs for some mycobacterial strains.

Intracellular localization of dapsone and rifampicin in skin and nerve of multidrug treated leprosy patients.

Intracellular localization of antileprosy drugs dapsone (DDS) and rifampicin (RFP) was carried out on skin and nerve lesions obtained from multidrug treated, multi (BL-LL)- and pauci (BT-TT) bacillary cases of leprosy using immunocytochemical techniques. Intracellular localization of the above drugs especially in macrophages and Schwann cells was aimed by using rabbit raised anti DDS and RFP polyclonal antibody in an indirect peroxidase assay. Our study records both intra and extracellular staining with anti DDS and RFP antibodies in the skin as well as nerve lesions of MB and PB cases treated with MDT. All the nerves under investigation had moderate to severe pathology and hence free diffusion of the drug could be attributed to the broken barrier. Basal lamina around the Schwann cell did not seem to form a barrier. It was also noted that the drug (metabolite) persisted over a long period of time).

Determination of rifampicin, desacetylrifampicin, isoniazid and acetylisoniazid by high-performance liquid chromatography: application to human serum extracts, polymorphonucleocytes and alveolar macrophages.

A method for the determination of rifampicin, desacetylrifampicin, isoniazid, and acetylisoniazid by high-performance liquid chromatography and using the same extract of the same sample is reported. After protein precipitation and extraction of these antituberculous drugs, two reversed-phase chromatographies were necessary. The technique was applied to serum extracts, polymorphonucleocytes and alveolar macrophages from patients treated for tuberculosis.