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1.
Sci Rep ; 10(1): 18120, 2020 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-33093532

RESUMO

Rifampicin resistance is a major therapeutic challenge, particularly in tuberculosis, leprosy, P. aeruginosa and S. aureus infections, where it develops via missense mutations in gene rpoB. Previously we have highlighted that these mutations reduce protein affinities within the RNA polymerase complex, subsequently reducing nucleic acid affinity. Here, we have used these insights to develop a computational rifampicin resistance predictor capable of identifying resistant mutations even outside the well-defined rifampicin resistance determining region (RRDR), using clinical M. tuberculosis sequencing information. Our tool successfully identified up to 90.9% of M. tuberculosis rpoB variants correctly, with sensitivity of 92.2%, specificity of 83.6% and MCC of 0.69, outperforming the current gold-standard GeneXpert-MTB/RIF. We show our model can be translated to other clinically relevant organisms: M. leprae, P. aeruginosa and S. aureus, despite weak sequence identity. Our method was implemented as an interactive tool, SUSPECT-RIF (StrUctural Susceptibility PrEdiCTion for RIFampicin), freely available at https://biosig.unimelb.edu.au/suspect_rif/ .


Assuntos
Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Aprendizado de Máquina , Mutação de Sentido Incorreto , Mycobacterium leprae/genética , Mycobacterium tuberculosis/genética , Rifampina/farmacologia , Staphylococcus aureus/genética , Antituberculosos/farmacologia , Proteínas de Bactérias/química , Humanos , Hanseníase/tratamento farmacológico , Hanseníase/microbiologia , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia
2.
Int J Mol Sci ; 21(17)2020 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-32824985

RESUMO

The aim of this study is to examine the use of an inflammasome competitor as a preventative agent. Coronaviruses have zoonotic potential due to the adaptability of their S protein to bind receptors of other species, most notably demonstrated by SARS-CoV. The binding of SARS-CoV-2 to TLR (Toll-like receptor) causes the release of pro-IL-1ß, which is cleaved by caspase-1, followed by the formation and activation of the inflammasome, which is a mediator of lung inflammation, fever, and fibrosis. The NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome is implicated in a variety of human diseases including Alzheimer's disease (AD), prion diseases, type 2 diabetes, and numerous infectious diseases. By examining the use of 4,4'-diaminodiphenyl sulfone (DDS) in the treatment of patients with Hansen's disease, also diagnosed as Alzheimer's disease, this study demonstrates the diverse mechanisms involved in the activation of inflammasomes. TLRs, due to genetic polymorphisms, can alter the immune response to a wide variety of microbial ligands, including viruses. In particular, TLR2Arg677Trp was reported to be exclusively present in Korean patients with lepromatous leprosy (LL). Previously, mutation of the intracellular domain of TLR2 has demonstrated its role in determining the susceptibility to LL, though LL was successfully treated using a combination of DDS with rifampicin and clofazimine. Of the three tested antibiotics, DDS was effective in the molecular regulation of NLRP3 inflammasome activators that are important in mild cognitive impairment (MCI), Parkinson's disease (PD), and AD. The specific targeting of NLRP3 itself or up-/downstream factors of the NLRP3 inflammasome by DDS may be responsible for its observed preventive effects, functioning as a competitor.


Assuntos
Infecções por Coronavirus/tratamento farmacológico , Dapsona/farmacologia , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Pneumonia Viral/tratamento farmacológico , Doença de Alzheimer/patologia , COVID-19 , Clofazimina/farmacologia , Disfunção Cognitiva/patologia , Humanos , Interleucina-1beta/metabolismo , Hanseníase/tratamento farmacológico , Hanseníase/genética , Pandemias , Transtornos Parkinsonianos/patologia , Rifampina/farmacologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Receptor 2 Toll-Like/genética
3.
Int J Mycobacteriol ; 9(2): 226-228, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32474551

RESUMO

Erythema multiforme (EM)-like erythema nodosum leprosum (ENL) is a rare atypical presentation, and its late appearance after the completion of multidrug therapy (MDT) is unusual. We describe the case of a lepromatous leprosy patient who after the completion of MDT presented to us with late EM-like ENL and was found to be resistant to rifampicin. We discuss the implications of this finding and the potential role of resistant bacilli in causing reactions with atypical presentations.


Assuntos
Quimioterapia Combinada/efeitos adversos , Hansenostáticos/uso terapêutico , Hanseníase/diagnóstico , Hanseníase/tratamento farmacológico , Rifampina/uso terapêutico , Adulto , Farmacorresistência Bacteriana/genética , Eritema Multiforme/diagnóstico , Eritema Multiforme/patologia , Eritema Nodoso/diagnóstico , Eritema Nodoso/patologia , Humanos , Masculino , Mycobacterium lepraemurium/efeitos dos fármacos , Mycobacterium lepraemurium/genética , Rifampina/farmacologia , Fatores de Tempo
4.
Artigo em Inglês | MEDLINE | ID: mdl-32340990

RESUMO

A case of Mycobacterium leprae rifampin resistance after irregular antileprosy treatments since 1971 is reported. Whole-genome sequencing from four longitudinal samples indicated relapse due to acquired rifampin resistance and not to reinfection with another strain. A putative compensatory mutation in rpoC was also detected. Clinical improvement was achieved using an alternative therapy.


Assuntos
Hanseníase , Mycobacterium leprae , Humanos , Hanseníase/tratamento farmacológico , Mutação , Mycobacterium leprae/genética , Recidiva , Rifampina/farmacologia
5.
Biochem Pharmacol ; 177: 113993, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32339493

RESUMO

Leprosy is a chronic infectious disease caused my Mycobacterium leprae that primarily affects peripheral nervous system and extremities and is prevalent in tropical countries. Treatment for leprosy with multidrug regimens is very effective compared to monotherapy especially in multibacillary cases. The three major antileprosy drugs currently in use are 4, 4'-diaminodiphenyl sulfone (DDS, dapsone), rifampicin, and clofazimine. During multidrug therapy, the potent antibiotic rifampicin induces the metabolism of dapsone, which results in decreased plasma half-life of dapsone and its metabolites. Furthermore, rifampicin induces its own metabolism and decreases its half-life during monotherapy. Rifampicin upregulates several hepatic microsomal drug-metabolizing enzymes, especially cytochrome P450 (CYP) family that in turn induce the metabolism of dapsone. Clofazimine lacks significant induction of any drug-metabolizing enzyme including CYP family and does not interact with dapsone metabolism. Rifampicin does not induce clofazimine metabolism during combination treatment. Administration of dapsone in the acetylated form (acedapsone) can release the drug slowly into circulation up to 75 days and could be useful for the effective treatment of paucibacillary cases along with rifampicin. This review summarizes the major aspects of antileprosy drug metabolism and drug interactions and the role of cytochrome P450 family of drug metabolizing enzymes, especially CYP3A4 during multidrug regimens for the treatment of leprosy.


Assuntos
Acedapsona/sangue , Clofazimina/sangue , Citocromo P-450 CYP3A/metabolismo , Dapsona/sangue , Hansenostáticos/sangue , Hanseníase/tratamento farmacológico , Rifampina/sangue , Acedapsona/farmacocinética , Acedapsona/farmacologia , Disponibilidade Biológica , Biotransformação , Clofazimina/farmacocinética , Clofazimina/farmacologia , Dapsona/farmacocinética , Dapsona/farmacologia , Interações Medicamentosas , Quimioterapia Combinada , Meia-Vida , Humanos , Hansenostáticos/farmacocinética , Hansenostáticos/farmacologia , Hanseníase/sangue , Hanseníase/microbiologia , Hanseníase/patologia , Taxa de Depuração Metabólica , Redes e Vias Metabólicas/fisiologia , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/crescimento & desenvolvimento , Mycobacterium leprae/patogenicidade , Rifampina/farmacocinética , Rifampina/farmacologia
6.
Molecules ; 25(3)2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-32046221

RESUMO

Therapeutic treatment options for opportunistic non-tuberculous mycobacterial (NTM) infection and/or serious mycobacterial infections such as tuberculosis (TB) and leprosy are limited due to the spread of antimicrobial resistance mechanism. Plant-derived natural compounds as prospective efflux pump inhibitors may present a promising adjunct to conventional chemotherapy by enhancing mycobacterial susceptibility to antibiotics. This study served to evaluate the antimicrobial and resistance-modifying profile of a range of plant-derived flavonoids against the mycobacterial model strains: M. smegmatis, M. aurum, and M. bovis BCG. The minimum inhibitory concentrations (MICs) of the compounds against the mycobacterial strains were determined using both agar dilution and broth dilution assays, while their efflux inhibitory activity was investigated via an ethidium bromide-based fluorometric assay. All compounds were screened for their synergistic effects with ethidium bromide (EtBr) and rifampicin (RIF) against M. smegmatis. Skullcapflavone II (5,2'-dihydroxy-6,7,8,6'-tetramethoxyflavone, 1) exerted potent antimicrobial activity against M. aurum and M. bovis BCG and considerably increased the susceptibility of M. smegmatis to EtBr and RIF. Nobiletin (5,6,7,8,3',4'-hexamethoxyflavone, 2) was determined to be the most potent efflux-inhibitor in M. aurum and M. smegmatis. However, a connection between strong modulatory and putative efflux activity of the compounds could not be observed. Nevertheless, the results highlight two polymethoxyflavones, skullcapflavone II and nobiletin, with potent antimycobacterial and antibiotic resistance modulating activities as valuable adjuvants in anti-mycobacterial therapies.


Assuntos
Anti-Infecciosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Flavonoides/farmacologia , Mycobacterium/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Transporte Biológico/efeitos dos fármacos , Etídio/química , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana/métodos , Mycobacterium/metabolismo , Infecções por Mycobacterium/tratamento farmacológico , Rifampina/farmacologia
7.
Indian J Med Res ; 152(5): 482-489, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33707390

RESUMO

Background & objectives: There is a need for an affordable, easy, high-sensitivity test usable at the peripheral health facility for diagnosis of drug-resistant (DR) tuberculosis (TB) to interrupt disease transmission. Nucleic acid amplification tests (NAATs) for early detection of DR-TB are ideal to bring testing near to the patient. TruenatTM MTB (Mycobacterium tuberculosis) and TruenatTM MTB-RIF (rifampicin) is an indigenous chip-based real-time polymerase chain reaction (PCR) based test for detection of multidrug-resistant (MDR) TB. The test involves extraction of DNA using automated, battery operated Trueprep instrument and real-time PCR performed on the Truelab analyzer. We report here multicentric validation of Truenat MTB-RIF for detection of DR-TB in suspected DR-TB patients. Methods: Consecutive patients aged 18-65 yr, with symptoms suggestive of TB and with a history of previous treatment, reporting to the National TB Elimination Programme (NTEP) clinics under four national institutes, namely AIIMS (All India Institute of Medical Sciences, New Delhi), NITRD (National Institute of Tuberculosis and Respiratory Diseases, New Delhi), NIRT (National Institute for Research in Tuberculosis, Chennai) and ICMR-National JALMA Institute for Leprosy and other Mycobacterial Diseases, Agra, were included in the study. Two sputum samples (one spot and one morning) were collected from each patient, after obtaining informed written consent. The samples were subjected to smear, GeneXpert and MGIT 960 culture (and drug susceptibility testing to RIF) (surrogate for MDR-TB) to serve as reference tests. The samples were coded to ensure blinding and subjected to Truenat MTB-RIF. Truenat MTB-RIF Version 1.5 was used for testing 1084 samples for RIF resistance, while Version 2.0 was used to test another 1201 samples. Results: Truenat MTB-RIF Version 1.5 in comparison with comprehensive laboratory reference standards yielded sensitivity and specificity of 76.2 and 94.7 per cent, respectively for the detection of RIF resistance in 1084 samples, collected across four sites. Based on the analysis of discordant samples, Version 2.0 of Truenat was developed by the manufacturer and this was further tested on additional 1201 samples, yielding a sensitivity of 87.5 per cent and specificity of 99.5 per cent. Interpretation & conclusions: Multicentric trial of TruenatTM MTB-RIF demonstrated a great potential of this point of care NAAT for detection of MDR-TB. The test would be useful in limited resource settings and inaccessible areas without need for any additional infrastructure.


Assuntos
Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Tuberculose Pulmonar , Adolescente , Adulto , Idoso , Humanos , Índia , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mycobacterium tuberculosis/genética , Rifampina/farmacologia , Rifampina/uso terapêutico , Sensibilidade e Especificidade , Escarro , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/genética , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/tratamento farmacológico , Adulto Jovem
8.
J Mater Chem B ; 7(42): 6539-6555, 2019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31584603

RESUMO

To unveil the effect of electrolyte concentration, pH and polymer addition on Tween 80 stabilized nanostructured lipid carriers (NLCs, based on dialkyldimethylammonium bromides DxDAB and Na oleate), an in-depth scattering analysis was performed. Dynamic and static light scattering (DLS/SLS) and small-angle neutron scattering (SANS) techniques along with zeta potential studies were exploited to understand the structural evolution and physical stability of NLCs. In these experiments, we varied the salt concentration, pH, and the admixture of Pluronic F127 in order to elucidate their effect on NLC morphologies. In most cases, two populations of different sizes are present which differ by one order of magnitude. The antileprosy drugs (ALD) Rifampicin and Dapsone were encapsulated in NLCs and the vector properties were assessed for a series of DxDAB (where x = 12, 14, 16 and 18) NLCs. The influence of composition on the entrapment and release behavior of NLCs was investigated: The size of NLCs correlates with the release rate of the incorporated drug. The interaction of drug-loaded NLCs with bovine serum albumin was studied to understand the release of ALD in the plasma.


Assuntos
Dapsona/farmacologia , Portadores de Fármacos/química , Hansenostáticos/farmacologia , Nanopartículas/química , Compostos de Amônio Quaternário/química , Rifampina/farmacologia , Animais , Bovinos , Portadores de Fármacos/metabolismo , Liberação Controlada de Fármacos , Cinética , Nanopartículas/metabolismo , Poloxâmero/química , Poloxâmero/metabolismo , Ligação Proteica , Compostos de Amônio Quaternário/metabolismo , Soroalbumina Bovina/metabolismo
9.
Colloids Surf B Biointerfaces ; 181: 845-855, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31254745

RESUMO

To date, the potential of sophorolipids (an important class of glycolipids) has been exploited solely as amphipathic molecules but their ability to formulate lipid nanoparticles has never been explored. In this report, for the first time, lipid nanostructures coated with polysorbates (Tweens) were formulated by a hot dispersion method. By varying the amount of lipid, type of surfactant, and alcohol, dilution ratio etc., the formulation was optimized with respect to its stability, which is a central aspect of their potential applications. Their comprehensive physicochemical characterization was done using static and dynamic light scattering (SLS, DLS), small angle neutron scattering (SANS), zeta-potential, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and atomic force microscopy (AFM) techniques. Further hemolysis study was conducted to understand the in-vitro cytotoxicity levels of the lipidic nanoparticles prior to its application as a potent drug delivery device for countermanding the problems associated with challenging tuberculosis and leprosy drug-Rifampicin. Attaining high entrapment efficiency and sustained release from the developed carrier, further interaction with bovine serum albumin was investigated, to understand the in-vivo behavior of the nanostructured lipid carriers (NLCs).


Assuntos
Materiais Biocompatíveis/química , Nanoestruturas/química , Ácidos Oleicos/química , Animais , Bovinos , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Eritrócitos/efeitos dos fármacos , Humanos , Tamanho da Partícula , Rifampina/química , Rifampina/farmacologia , Soroalbumina Bovina/química , Propriedades de Superfície
10.
Sci Rep ; 8(1): 5016, 2018 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-29567948

RESUMO

The rpoB gene encodes the ß subunit of RNA polymerase holoenzyme in Mycobacterium leprae (M. leprae). Missense mutations in the rpoB gene were identified as etiological factors for rifampin resistance in leprosy. In the present study, we identified mutations corresponding to rifampin resistance in relapsed leprosy cases from three hospitals in southern India which treat leprosy patients. DNA was extracted from skin biopsies of 35 relapse/multidrug therapy non-respondent leprosy cases, and PCR was performed to amplify the 276 bp rifampin resistance-determining region of the rpoB gene. PCR products were sequenced, and mutations were identified in four out of the 35 cases at codon positions D441Y, D441V, S437L and H476R. The structural and functional effects of these mutations were assessed in the context of three-dimensional comparative models of wild-type and mutant M. leprae RNA polymerase holoenzyme (RNAP), based on the recently solved crystal structures of RNAP of Mycobacterium tuberculosis, containing a synthetic nucleic acid scaffold and rifampin. The resistance mutations were observed to alter the hydrogen-bonding and hydrophobic interactions of rifampin and the 5' ribonucleotide of the growing RNA transcript. This study demonstrates that rifampin-resistant strains of M. leprae among leprosy patients in southern India are likely to arise from mutations that affect the drug-binding site and stability of RNAP.


Assuntos
Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/genética , Farmacorresistência Bacteriana/genética , Hansenostáticos/farmacologia , Hanseníase/tratamento farmacológico , Mycobacterium leprae/genética , Rifampina/farmacologia , Adolescente , Adulto , DNA Bacteriano/genética , Feminino , Humanos , Índia , Hansenostáticos/uso terapêutico , Hanseníase/microbiologia , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mutação , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/isolamento & purificação , Ligação Proteica/genética , Estabilidade Proteica/efeitos dos fármacos , Recidiva , Rifampina/uso terapêutico , Análise de Sequência de DNA , Relação Estrutura-Atividade , Resultado do Tratamento , Adulto Jovem
11.
PLoS Negl Trop Dis ; 11(6): e0005506, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28570560

RESUMO

BACKGROUND: Real-Time PCR-High Resolution Melting (qPCR-HRM) analysis has been recently described for rapid drug susceptibility testing (DST) of Mycobacterium leprae. The purpose of the current study was to further evaluate the validity, reliability, and accuracy of this assay for M. leprae DST in clinical specimens. METHODOLOGY/PRINCIPAL FINDINGS: The specificity and sensitivity for determining the presence and susceptibility of M. leprae to dapsone based on the folP1 drug resistance determining region (DRDR), rifampin (rpoB DRDR) and ofloxacin (gyrA DRDR) was evaluated using 211 clinical specimens from leprosy patients, including 156 multibacillary (MB) and 55 paucibacillary (PB) cases. When comparing the results of qPCR-HRM DST and PCR/direct DNA sequencing, 100% concordance was obtained. The effects of in-house phenol/chloroform extraction versus column-based DNA purification protocols, and that of storage and fixation protocols of specimens for qPCR-HRM DST, were also evaluated. qPCR-HRM results for all DRDR gene assays (folP1, rpoB, and gyrA) were obtained from both MB (154/156; 98.7%) and PB (35/55; 63.3%) patients. All PCR negative specimens were from patients with low numbers of bacilli enumerated by an M. leprae-specific qPCR. We observed that frozen and formalin-fixed paraffin embedded (FFPE) tissues or archival Fite's stained slides were suitable for HRM analysis. Among 20 mycobacterial and other skin bacterial species tested, only M. lepromatosis, highly related to M. leprae, generated amplicons in the qPCR-HRM DST assay for folP1 and rpoB DRDR targets. Both DNA purification protocols tested were efficient in recovering DNA suitable for HRM analysis. However, 3% of clinical specimens purified using the phenol/chloroform DNA purification protocol gave false drug resistant data. DNA obtained from freshly frozen (n = 172), formalin-fixed paraffin embedded (FFPE) tissues (n = 36) or archival Fite's stained slides (n = 3) were suitable for qPCR-HRM DST analysis. The HRM-based assay was also able to identify mixed infections of susceptible and resistant M. leprae. However, to avoid false positives we recommend that clinical specimens be tested for the presence of the M. leprae using the qPCR-RLEP assay prior to being tested in the qPCR-HRM DST and that all specimens demonstrating drug resistant profiles in this assay be subjected to DNA sequencing. CONCLUSION/SIGNIFICANCE: Taken together these results further demonstrate the utility of qPCR-HRM DST as an inexpensive screening tool for large-scale drug resistance surveillance in leprosy.


Assuntos
Farmacorresistência Bacteriana/genética , Hanseníase/tratamento farmacológico , Testes de Sensibilidade Microbiana/métodos , Mycobacterium leprae/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Dapsona/farmacologia , Humanos , Hansenostáticos/farmacologia , Hanseníase/microbiologia , Mycobacterium leprae/isolamento & purificação , Ofloxacino/farmacologia , Reprodutibilidade dos Testes , Rifampina/farmacologia , Sensibilidade e Especificidade , Análise de Sequência de DNA , Pele/microbiologia , Pele/patologia
12.
Clin Microbiol Infect ; 23(3): 167-172, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27664776

RESUMO

The rpoB gene codes for the RNA polymerase ß subunit, which is the target of rifampicin, an essential drug in the treatment of tuberculosis and other mycobacterial infections. This gene is present in all bacteria, but its length and nucleotide sequence vary between bacterial species, including mycobacteria. Mutations in the rpoB gene alter the structure of this protein and cause drug resistance. To describe the resistance-associated mutations, the scientific and medical communities have been using, since 1993, a numbering system based on the Escherichia coli sequence annotation. Using E. coli reference for describing mutations in mycobacteria leads to misunderstandings, particularly with the increasing use of whole genome sequencing, which brought an alternative numbering system based on the Mycobacterium tuberculosis rpoB sequence. We propose using a consensus numbering system for the reporting of resistance mutations based on the reference genomes from the species interrogated (such as strain H37Rv for M. tuberculosis). This manuscript provides the necessary figures and tables allowing researchers, microbiologists and clinicians to easily convert other annotation systems into one common language.


Assuntos
Antibióticos Antituberculose/farmacologia , Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/genética , Genótipo , Técnicas de Genotipagem/normas , Testes de Sensibilidade Microbiana/normas , Proteínas Mutantes/genética , Rifampina/farmacologia , Consenso , Escherichia coli , Proteínas de Escherichia coli/genética , Humanos , Mutação , Mycobacterium/efeitos dos fármacos , Mycobacterium tuberculosis , Terminologia como Assunto
13.
s.l; s.n; 2017. 18 p. tab, graf.
Não convencional em Inglês | SES-SP, HANSEN, SESSP-ILSLPROD, SES-SP, SESSP-ILSLACERVO, SES-SP | ID: biblio-1053286

RESUMO

BACKGROUND: Real-Time PCR-High Resolution Melting (qPCR-HRM) analysis has been recently described for rapid drug susceptibility testing (DST) of Mycobacterium leprae. The purpose of the current study was to further evaluate the validity, reliability, and accuracy of this assay for M. leprae DST in clinical specimens. METHODOLOGY/PRINCIPAL FINDINGS: The specificity and sensitivity for determining the presence and susceptibility of M. leprae to dapsone based on the folP1 drug resistance determining region (DRDR), rifampin (rpoB DRDR) and ofloxacin (gyrA DRDR) was evaluated using 211 clinical specimens from leprosy patients, including 156 multibacillary (MB) and 55 paucibacillary (PB) cases. When comparing the results of qPCR-HRM DST and PCR/direct DNA sequencing, 100% concordance was obtained. The effects of in-house phenol/chloroform extraction versus column-based DNA purification protocols, and that of storage and fixation protocols of specimens for qPCR-HRM DST, were also evaluated. qPCR-HRM results for all DRDR gene assays (folP1, rpoB, and gyrA) were obtained from both MB (154/156; 98.7%) and PB (35/55; 63.3%) patients. All PCR negative specimens were from patients with low numbers of bacilli enumerated by an M. leprae-specific qPCR. We observed that frozen and formalin-fixed paraffin embedded (FFPE) tissues or archival Fite's stained slides were suitable for HRM analysis. Among 20 mycobacterial and other skin bacterial species tested, only M. lepromatosis, highly related to M. leprae, generated amplicons in the qPCR-HRM DST assay for folP1 and rpoB DRDR targets. Both DNA purification protocols tested were efficient in recovering DNA suitable for HRM analysis. However, 3% of clinical specimens purified using the phenol/chloroform DNA purification protocol gave false drug resistant data. DNA obtained from freshly frozen (n = 172), formalin-fixed paraffin embedded (FFPE) tissues (n = 36) or archival Fite's stained slides (n = 3) were suitable for qPCR-HRM DST analysis. The HRM-based assay was also able to identify mixed infections of susceptible and resistant M. leprae. However, to avoid false positives we recommend that clinical specimens be tested for the presence of the M. leprae using the qPCR-RLEP assay prior to being tested in the qPCR-HRM DST and that all specimens demonstrating drug resistant profiles in this assay be subjected to DNA sequencing. CONCLUSION/SIGNIFICANCE: Taken together these results further demonstrate the utility of qPCR-HRM DST as an inexpensive screening tool for large-scale drug resistance surveillance in leprosy.


Assuntos
Humanos , Rifampina/farmacologia , Pele/microbiologia , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Ofloxacino/farmacologia , Testes de Sensibilidade Microbiana/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNA , Farmacorresistência Bacteriana/genética , Dapsona/farmacologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Hansenostáticos/farmacologia , Hanseníase/microbiologia , Hanseníase/tratamento farmacológico , Mycobacterium leprae/isolamento & purificação , Mycobacterium leprae/efeitos dos fármacos
14.
PLoS Negl Trop Dis ; 10(10): e0005041, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27706165

RESUMO

An active search for Mycobacterium leprae drug resistance was carried out, 243 multibacillary patients from endemic regions of Colombia were included from 2004 to 2013 in a surveillance program. This program was a World Health Organization initiative for drug resistance surveillance in leprosy, where Colombia is a sentinel country. M. leprae DNA from slit skin smear and/or skin biopsy samples was amplified and sequenced to identify mutations in the drug resistance determining region (DRDR) in rpoB, folP1, gyrA, and gyrB, the genes responsible for rifampicin, dapsone and ofloxacin drug-resistance, respectively. Three isolates exhibited mutations in the DRDR rpoB gene (Asp441Tyr, Ser456Leu, Ser458Met), two in the DRDR folP1 gene (Thr53Ala, Pro55Leu), and one isolate exhibited mutations in both DRDR rpoB (Ser456Met) and DRDR folP1 (Pro55Leu), suggesting multidrug resistance. One isolate had a double mutation in folP1 (Thr53Ala and Thr88Pro). Also, we detected mutations outside of DRDR that required in vivo evaluation of their association or not with drug resistance: rpoB Arg505Trp, folP1 Asp91His, Arg94Trp, and Thr88Pro, and gyrA Ala107Leu. Seventy percent of M. leprae mutations were related to drug resistance and were isolated from relapsed patients; the likelihood of relapse was significantly associated with the presence of confirmed resistance mutations (OR range 20.1-88.7, p < 0.05). Five of these relapsed patients received dapsone monotherapy as a primary treatment. In summary, the current study calls attention to M. leprae resistance in Colombia, especially the significant association between confirmed resistance mutations and relapse in leprosy patients. A high frequency of DRDR mutations for rifampicin was seen in a region where dapsone monotherapy was used extensively.


Assuntos
Proteínas de Bactérias/genética , Farmacorresistência Bacteriana , Hansenostáticos/farmacologia , Hanseníase/microbiologia , Mycobacterium leprae/efeitos dos fármacos , Vigilância de Evento Sentinela , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Colômbia/epidemiologia , DNA Bacteriano/genética , Dapsona/farmacologia , Dapsona/uso terapêutico , Farmacorresistência Bacteriana/genética , Farmacorresistência Bacteriana Múltipla , Feminino , Humanos , Hansenostáticos/uso terapêutico , Hanseníase/tratamento farmacológico , Hanseníase/epidemiologia , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mutação , Mycobacterium leprae/genética , Mycobacterium leprae/isolamento & purificação , Ofloxacino/farmacologia , Ofloxacino/uso terapêutico , Reação em Cadeia da Polimerase , Recidiva , Rifampina/farmacologia , Rifampina/uso terapêutico , Pele/microbiologia , Adulto Jovem
15.
Clin Infect Dis ; 63(11): 1482-1484, 2016 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-27558568

RESUMO

Molecular drug susceptibility testing was performed on skin biopsies from 24 leprosy patients from Guinea-Conakry for the first time. We identified primary drug resistance in 4 cases and a dapsone-resistant cluster caused by the same strain. Primary transmission of drug-resistant Mycobacterium leprae, including a rifampicin-resistant strain, is reported.


Assuntos
Antibióticos Antituberculose/farmacologia , Antituberculosos/farmacologia , Resistência Microbiana a Medicamentos , Hanseníase/microbiologia , Hanseníase/transmissão , Mycobacterium leprae/efeitos dos fármacos , Antibióticos Antituberculose/uso terapêutico , Antituberculosos/uso terapêutico , Biópsia , DNA Bacteriano/genética , Dapsona/farmacologia , Dapsona/uso terapêutico , Feminino , Genoma Bacteriano , Guiné/epidemiologia , Humanos , Hanseníase/epidemiologia , Masculino , Mycobacterium leprae/genética , Mycobacterium leprae/isolamento & purificação , Rifampina/farmacologia , Rifampina/uso terapêutico , Análise de Sequência de DNA , Pele/microbiologia , Pele/patologia
16.
Infect Dis Poverty ; 5(1): 46, 2016 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-27268059

RESUMO

Post-exposure prophylaxis (PEP) for leprosy is administered as one single dose of rifampicin (SDR) to the contacts of newly diagnosed leprosy patients. SDR reduces the risk of developing leprosy among contacts by around 60 % in the first 2-3 years after receiving SDR. In countries where SDR is currently being implemented under routine programme conditions in defined areas, questions were raised by health authorities and professional bodies about the possible risk of inducing rifampicin resistance among the M. tuberculosis strains circulating in these areas. This issue has not been addressed in scientific literature to date. To produce an authoritative consensus statement about the risk that SDR would induce rifampicin-resistant tuberculosis, a meeting was convened with tuberculosis (TB) and leprosy experts. The experts carefully reviewed and discussed the available evidence regarding the mechanisms and risk factors for the development of (multi) drug-resistance in M. tuberculosis with a view to the special situation of the use of SDR as PEP for leprosy. They concluded that SDR given to contacts of leprosy patients, in the absence of symptoms of active TB, poses a negligible risk of generating resistance in M. tuberculosis in individuals and at the population level. Thus, the benefits of SDR prophylaxis in reducing the risk of developing leprosy in contacts of new leprosy patients far outweigh the risks of generating drug resistance in M. tuberculosis.


Assuntos
Farmacorresistência Bacteriana , Hansenostáticos/uso terapêutico , Hanseníase/tratamento farmacológico , Mycobacterium tuberculosis/efeitos dos fármacos , Profilaxia Pós-Exposição , Rifampina/farmacologia , Rifampina/uso terapêutico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Humanos , Hansenostáticos/farmacologia , Risco
17.
Lepr Rev ; 87(1): 93-100, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27255062

RESUMO

BACKGROUND: Rifampicin is the major drug in the treatment of leprosy. The rifampicin resistance of Mycobacterium leprae results from a mutation in the rpoB gene, encoding the ß subunit of RNA polymerase. As M. leprae is a non-cultivable organism observation of its growth using mouse food-pad (MFP) is the only Gold Standard assay used for confirmation of "in-vivo" drug resistance. OBJECTIVE: Any mutation at molecular level has to be verified by MFP assay for final confirmation of drug resistance in leprosy. MATERIAL AND METHODS: In the present study, M. leprae strains showing a mutation only at codon 442 Gln-His and along with mutation either at codon 424 Val-Gly or at 438 Gln-Val within the Rifampicin Resistance Determining Region (RRDR) confirmed by DNA sequencing and by high resolution melting (HRM) analysis were subjected for its growth in MFP. RESULT AND CONCLUSION: The M. leprae strain having the new mutation at codon 442 Gln-His was found to be sensitive to all the three drugs and strains having additional mutations at 424 Val-Gly and 438 Gln-Val were conferring resistance with Multi drug therapy (MDT) in MFP. These results indicate that MFP is the gold standard method for confirming the mutations detected by molecular techniques.


Assuntos
Proteínas de Bactérias/metabolismo , Hansenostáticos/farmacologia , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/genética , Rifampina/farmacologia , Adulto , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Bioensaio , DNA Bacteriano , Regulação Bacteriana da Expressão Gênica/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Reação em Cadeia da Polimerase , Adulto Jovem
18.
J Microbiol ; 53(10): 686-93, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26428919

RESUMO

Drug resistance in Mycobacterium leprae is a significant problem in countries where leprosy is endemic. A sensitive, specific, and high-throughput reverse blot hybridization assay (REBA) for the detection of genotypic resistance to rifampicin (RIF) was designed and evaluated. It has been shown that resistance to RIF in M. leprae involves mutations in the rpoB gene encoding the -subunit of the RNA polymerase. The PCR-REBA simultaneously detects both 6 wild-type regions and 5 different mutations (507 AGC, 513 GTG, 516 TAT, 531 ATG, and 531 TTC) including the most prevalent mutations at positions 507 and 531. Thirty-one clinical isolates provided by Korea Institute of Hansen-s Disease were analyzed by PCR-REBA with RIF resistance of rpoB gene. As a result, missense mutations at codons 507 AGC and 531 ATG with 2-nucleotide substitutions were found in one sample, and a missense mutation at codon 516 TAT and ΔWT6 (deletion of 530-534) was found in another sample. These cases were confirmed by DNA sequence analysis. This rapid, simple, and highly sensitive assay provides a practical alternative to sequencing for genotypic evaluation of RIF resistance in M. leprae.


Assuntos
Antibióticos Antituberculose/farmacologia , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/genética , Hibridização de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Rifampina/farmacologia , Sequência de Aminoácidos , Sequência de Bases , RNA Polimerases Dirigidas por DNA/genética , Farmacorresistência Bacteriana , Humanos , Dados de Sequência Molecular , Mutação , Mycobacterium leprae/isolamento & purificação , Sensibilidade e Especificidade
19.
Drug Metab Pers Ther ; 30(4): 257-61, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26353177

RESUMO

BACKGROUND: Increased exsorption of domperidone was observed from different parts of the small intestine of the rat after pretreatment with rifampicin by the everted sac method. Based on the in vitro studies the effect of rifampicin pretreatment on the pharmacokinetics of domperidone was investigated in eight healthy male volunteers. METHODS: After an overnight fast, 20 mg domperidone was administered to the volunteers, either alone or after 6 days pretreatment with a once daily dose of 600 mg rifampicin. Serum concentrations of domperidone were estimated by reverse phase HPLC. Pharmacokinetic parameters were determined based on non-compartmental model analysis using the computer program kinetica. RESULTS: Rifampicin pretreatment decreased Cmax, AUCo-∞, AUMC, MRT and t1/2 by 25.11%, 37.76%, 64.97%, 43.71% and 44.48%, respectively. This may be due to increased induction of cytochrome P450 enzymes and/or increased expression of P-glycoprotein. CONCLUSIONS: This interaction may have clinical significance when domperidone is co-administered with rifampicin in chronic treatment conditions, such as tuberculosis, leprosy and other infections of joints, bones, etc.


Assuntos
Indutores do Citocromo P-450 CYP3A/farmacologia , Domperidona/farmacocinética , Antagonistas de Dopamina/farmacocinética , Rifampina/farmacologia , Adulto , Análise de Variância , Animais , Disponibilidade Biológica , Combinação de Medicamentos , Voluntários Saudáveis , Humanos , Intestino Delgado/metabolismo , Masculino , Ratos Wistar
20.
Clin Exp Dermatol ; 40(8): 908-11, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25991507

RESUMO

BACKGROUND: Previous studies of drug resistance have shown that mutations in the drug resistance-determining region (DRDR) in the Folp1, RpoB and GyrA genes of Mycobacterium leprae are responsible for resistance to dapsone, rifampin and ofloxacin, respectively. AIM: To investigate the prevalence of mutations in genes associated with drug resistance in M. leprae isolates from patients with leprosy in Shandong Province. METHODS: The DRDR in the FolP1, RpoB and GyrA genes was analysed by direct sequencing of the PCR product from 85 isolates of M. leprae sampled from patients with leprosy in Shandong, China. RESULTS: Sequencing results were obtained for FolP1, RpoB and GyrA in 67, 57 and 81 of the 85 samples, with mutation rates of 1.5% (1/67), 8.8% 5/57 and 25.9% (21/81). Three multidrug-resistant samples were found among the new cases: one had a mutation in both Folp1 and RpoB, while the other two had a mutation in both RpoB and GyrA. CONCLUSIONS: Primary resistance appears to be to either single drugs or combinations of two drugs. The resistance rate to dapsone seems to be low. To our knowledge, this is the first case of multidrug-resistant M. leprae from China.


Assuntos
Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Hanseníase/microbiologia , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/genética , Adulto , China/epidemiologia , DNA Bacteriano/genética , Dapsona/farmacologia , Feminino , Humanos , Hansenostáticos/farmacologia , Hanseníase/epidemiologia , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mutação , Ofloxacino/farmacologia , Reação em Cadeia da Polimerase , Prevalência , Rifampina/farmacologia
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