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1.
Diagn Microbiol Infect Dis ; 100(1): 115325, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33556650

RESUMO

Although multidrug therapy is considered an effective treatment for leprosy, antimicrobial resistance is a serious concern. We performed a systematic review of studies on the diagnostic accuracy and screening of tests for antimicrobial resistance in leprosy. This review was registered in PROSPERO (CRD42020177958). In April 2020, we searched for studies in the PubMed, EMBASE, Web of Science, Scopus, Scielo, and LILACS databases. A random effects regression model was used for the meta-analysis. We included 129 studies. Molecular tests for dapsone resistance had a sensitivity of 78.8% (95% confidence interval [CI] = 65.6-87.9) and a specificity of 97.0% (95% CI = 94.0-98.6). Molecular tests for rifampicin resistance had a sensitivity and specificity of 88.7% (95% CI = 80.0-93.9) and 97.3% (95% CI = 94.3-98.8), respectively. Molecular tests for ofloxacin resistance had a sensitivity and specificity of 80.9% (95% CI = 60.1-92.3) and 96.1% (95% CI = 90.2-98.5), respectively. In recent decades, no increase in the resistance proportion was detected. However, the growing number of resistant cases is still a clinical concern.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Hanseníase , Testes de Sensibilidade Microbiana , Mycobacterium leprae/efeitos dos fármacos , DNA Bacteriano/genética , Humanos , Hanseníase/diagnóstico , Hanseníase/microbiologia , Testes de Sensibilidade Microbiana/métodos , Testes de Sensibilidade Microbiana/normas , Sensibilidade e Especificidade , Análise de Sequência de DNA
2.
Artigo em Inglês | MEDLINE | ID: mdl-33361310

RESUMO

Mycobacteroides abscessus (Mab) is an opportunistic environmental pathogen that can cause chronic pulmonary disease in the setting of structural lung conditions such as bronchiectasis, chronic obstructive pulmonary disease, and cystic fibrosis. These infections are often incurable and associated with rapid lung function decline. Mab is naturally resistant to most of the antibiotics available today, and current treatment guidelines require at least 1 year of daily multidrug therapy, which is often ineffective and is associated with significant toxicities. ß-Lactams are the most widely used class of antibiotics and have a demonstrated record of safety and tolerability. Here, using a panel of recent clinical isolates of Mab, we evaluated the in vitro activities of dual-ß-lactam combinations to identify new treatments with the potential to treat infections arising from a wide range of Mab strains. The Mab clinical isolates were heterogeneous, as reflected by the diversity of their genomes and differences in their susceptibilities to various drugs. Cefoxitin and imipenem are currently the only two ß-lactams included in the guidelines for treating Mab disease, yet they are not used concurrently in clinical practice. However, this dual-ß-lactam combination exhibited synergy against 100% of the isolates examined (n = 21). Equally surprising is the finding that the combination of two carbapenems, doripenem and imipenem, exhibited synergy against the majority of Mab isolates. In the setting of multidrug-resistant Mab disease with few therapeutic options, these combinations may offer viable immediate treatment options with efficacy against the broad spectrum of Mab strains infecting patients today.


Assuntos
Mycobacterium abscessus , Antibacterianos/farmacologia , Quimioterapia Combinada , Humanos , Hansenostáticos , Testes de Sensibilidade Microbiana , beta-Lactamas/farmacologia
3.
Int J Mycobacteriol ; 9(1): 34-38, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32474486

RESUMO

Background: Fluoroquinolones (FQs) are being used as second-line agents in the treatment of tuberculosis caused by multidrug-resistant strains. Ofloxacin (OFX) is being tried as a part of modified multidrug therapy regimens for leprosy. A preliminary study was carried out to evaluate the accumulation of FQs - OFX, levofloxacin (LFX), norfloxacin (NFX), and ciprofloxacin (CIF) in Mycobacterium smegmatis. Methods: M. smegmatis were grown in Sauton's medium till log phase, harvested and resuspended in phosphate buffer (0.1 M, pH 7.2, Optical Density (OD) of 0.4-0.5) The suspensions were incubated with OFX, LFX, NFX, and CIF (10 µg/ml) at 37°C. The drugs were estimated in the supernatants using spectrofluorimeteric methods. The experiments were also conducted with the addition of carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a proton motive force inhibitor, at 100 µM, 10 min before and/or immediately after the addition of the drugs. Results: The time taken to achieve a Steady State Concentration (SSC) of OFX in M. smegmatis was 3 min and the level of accumulation was 102 ng/mg dry weight of the bacilli; with LFX the time for SSC was 5 min and the level of accumulation was 90 ng/mg; in case of NFX the accumulation to SSC was 87 ng/mg in 3 min. CIF accumulation attained a steady state (SSC level of 79 ng/mg) in 4 min. The accumulation kinetics for NFX in M. smegmatis using the spectrofluorimetric method is comparable with radioactive assays. Dose-related accumulation was observed with 10 µg/ml exposure concentrations. The addition of CCCP failed to influence the accumulation of each of these quinolones. Conclusion: The findings of dose-related accumulation of OFX, LFX NFX, and CIF suggest simple diffusion as the possible mechanism of transport of these drugs.


Assuntos
Fluorometria/métodos , Fluoroquinolonas/farmacocinética , Hansenostáticos/farmacocinética , Mycobacterium smegmatis/metabolismo , Permeabilidade da Membrana Celular , Meios de Cultura/química , Testes de Sensibilidade Microbiana
4.
PLoS One ; 15(4): e0231320, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32267877

RESUMO

INTRODUCTION: Tuberculosis (TB) remains a major global public health problem and is the leading cause of death from a single bacterium, Mycobacterium tuberculosis (MTB) complex. The emergence and spread of drug-resistant strains aggravate the problem, especially in tuberculosis high burden countries such as Ethiopia. The supposedly high initial cost of laboratory diagnosis coupled with scarce financial resources has limited collection of information about drug resistance patterns and circulating strains in peripheral and emerging regions of Ethiopia. Here, we investigated drug susceptibility and genetic diversity of mycobacterial isolates among pulmonary tuberculosis patients in the Benishangul Gumuz region and its surroundings in northwest Ethiopia. METHODS AND MATERIAL: In a cross-sectional study, 107 consecutive sputum smear-positive pulmonary tuberculosis (PTB) patients diagnosed at two hospitals and seven health centers were enrolled between October 2013 and June 2014. Sputum samples were cultured at Armauer Hansen Research Institute (AHRI) TB laboratory, and drug susceptibility testing (DST) was performed against Isoniazid, Rifampicin, Ethambutol, and Streptomycin using the indirect proportion method. Isolates were characterized using polymerase chain reaction (PCR)based Region of Difference 9 (RD9) testing and spoligotyping. Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) for Windows version 24.0. RESULTS: Of 107 acid-fast-bacilli (AFB) smear-positive sputum samples collected, 81.3% (87/107) were culture positive. A PCR based RD9 testing revealed that all the 87 isolates were M. tuberculosis. Of these isolates, 16.1% (14/87) resistance to one or more drugs was observed. Isoniazid monoresistance occurred in 6.9% (6/87). Multidrug resistance (MDR) was observed in two isolates (2.3%), one of which was resistant to all the four drugs tested. Spoligotyping revealed that the majority, 61.3% (46/75) of strains could be grouped into ten spoligotype patterns containing two to 11 isolates each while the remaining 38.7% (29/75) were unique. SIT289 (11 isolates) and SIT53 (nine isolates) constituted 43.5% (20/46) among clustered isolates while 29.3% (22/75) were ''New" to the database. The dominant families were T, 37% (28/75), CAS, 16.0% (12/75), and H, 8% (6/75), adding up to 51.3% (46/75) of all isolates identified. CONCLUSION AND RECOMMENDATIONS: The current study indicates a moderate prevalence of MDR TB. However, the observed high monoresistance to Isoniazid, one of the two proxy drugs for MDR-TB, reveals the hidden potential threat fora sudden increase in MDR-TB if resistance to Rifampicin would increase. Clustered spoligotype patterns suggest ongoing active tuberculosis transmission in the area. The results underscore the need for enhanced monitoring of TB drug resistance and epidemiological studies in this and other peripheral regions of the country using robust molecular tools with high discriminatory power such as the Mycobacterial Interspersed Repetitive Units -Variable Number of Tandem Repeats (MIRU-VNTR) typing and whole-genome sequencing (WGS).


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Variação Genética , Mycobacterium tuberculosis/genética , Tuberculose Pulmonar/diagnóstico , Adolescente , Adulto , Idoso , Antituberculosos/farmacologia , Estudos Transversais , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Etambutol/farmacologia , Etiópia/epidemiologia , Feminino , Humanos , Isoniazida/farmacologia , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Tipagem de Sequências Multilocus , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Escarro/microbiologia , Tuberculose Pulmonar/epidemiologia , Tuberculose Pulmonar/microbiologia , Adulto Jovem
5.
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
6.
Emerg Infect Dis ; 26(3): 615-617, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32091380

RESUMO

Using shotgun metagenomics, we identified an imported case of multidrug-resistant Mycobacterium leprae in a Filipino resident of Saudi Arabia in 2017. We determined the phylogenomic lineage (3K1) and identified mutations in rpoB and rrs corresponding to the multidrug-resistance phenotype clinically observed. Metagenomics sequencing can be used to identify multidrug-resistant M. leprae.


Assuntos
Hanseníase/diagnóstico , Mycobacterium leprae/isolamento & purificação , Adulto , DNA Bacteriano/genética , Farmacorresistência Bacteriana , Emigrantes e Imigrantes , Feminino , Humanos , Hansenostáticos/farmacologia , Hansenostáticos/uso terapêutico , Hanseníase/tratamento farmacológico , Metagenômica , Testes de Sensibilidade Microbiana , Mycobacterium leprae/efeitos dos fármacos , Filipinas/etnologia , Arábia Saudita
8.
Am J Trop Med Hyg ; 102(3): 547-552, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31933458

RESUMO

Resistance to anti-leprosy drugs is on the rise. Several studies have documented resistance to rifampicin, dapsone, and ofloxacin in patients with leprosy. We looked for point mutations within the folP1, rpoB, and gyrA gene regions of the Mycobacterium leprae genome predominantly in the neural form of leprosy. DNA samples from 77 nerve tissue samples were polymerase chain reaction (PCR)-amplified for M leprae DNA and sequenced for drug resistance-determining regions of genes rpoB, folP1, and gyrA. The mean age at presentation and onset was 38.2 ± 13.4 (range 14-71) years and 34.9 ± 12.6 years (range 10-63) years, respectively. The majority had borderline tuberculoid leprosy (53 [68.8%]). Mutations associated with resistance were identified in 6/77 (7.8%) specimens. Mutations seen were those associated with resistance to rifampicin, ofloxacin, and dapsone. All the six patients were drug-naive. The clinical and pathological manifestations in this group did not differ from the drug-sensitive group. This study highlights the occurrence of resistance to the standard multidrug therapy and ofloxacin in leprosy. Among the entire cohort, 1/77 (1.3%) showed resistance to rifampicin, 2/77 (2.6%) to dapsone, and 5/77 (6.4%) to ofloxacin. Six new patients showing infection by mutant strains indicated the emergence of primary resistance. Resistance to ofloxacin could be due to frequent use of quinolones for many bacterial infections. The results of the study indicate the need for development of a robust and strict surveillance system for detecting drug resistance in leprosy in India.


Assuntos
Hansenostáticos/uso terapêutico , Hanseníase/tratamento farmacológico , Hanseníase/microbiologia , Mycobacterium leprae/efeitos dos fármacos , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Estudos de Coortes , Estudos Transversais , Farmacorresistência Bacteriana , Quimioterapia Combinada , Feminino , Humanos , Índia/epidemiologia , Lactente , Hansenostáticos/farmacologia , Hanseníase/epidemiologia , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mutação , Mycobacterium leprae/genética , Adulto Jovem
9.
Clin Infect Dis ; 70(10): 2054-2061, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-31260522

RESUMO

BACKGROUND: Leprosy has been treated with multidrug therapy, which has been distributed for free across the globe and regarded as highly efficient. However, the impossibility of growing Mycobacterium leprae in axenic media has historically impaired assessments of M. leprae resistance, a parameter only recently detectable through molecular methods. METHODS: A systematic, population-based search for M. leprae resistance in suspected leprosy relapse cases and contacts was performed in Prata Village, an isolated, hyperendemic, former leprosy colony located in the Brazilian Amazon. Results led to an extended active search involving the entire Prata population. Confirmed leprosy cases were investigated for bacterial resistance using a combination of in vivo testing and direct sequencing of resistance genes folP1, rpoB, and gyrA. A molecular epidemiology analysis was performed using data from 17 variable number tandem repeats (VNTR). RESULTS: Mycobacterium leprae was obtained from biopsies of 37 leprosy cases (18 relapses and 19 new cases): 16 (43.24%) displayed drug-resistance variants. Multidrug resistance to rifampicin and dapsone was observed in 8 relapses and 4 new cases. Single resistance to rifampicin was detected in 1 new case. Resistance to dapsone was present in 2 relapses and 1 new case. Combined molecular resistance and VNTR data revealed evidence of intra-familial primary transmission of resistant M. leprae. CONCLUSIONS: A comprehensive, population-based systematic approach to investigate M. leprae resistance in a unique population revealed an alarming scenario of the emergence and transmission of resistant strains. These findings may be used for the development of new strategies for surveillance of drug resistance in other populations.


Assuntos
Hanseníase , Preparações Farmacêuticas , Brasil/epidemiologia , Farmacorresistência Bacteriana , Quimioterapia Combinada , Humanos , Hansenostáticos/farmacologia , Hansenostáticos/uso terapêutico , Hanseníase/tratamento farmacológico , Hanseníase/epidemiologia , Testes de Sensibilidade Microbiana , Mycobacterium leprae/genética
10.
J Antimicrob Chemother ; 75(3): 609-617, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31886864

RESUMO

BACKGROUND: Pulmonary infections caused by non-tuberculous mycobacteria (NTM) are hard to treat and have low cure rates despite intensive multidrug therapy. OBJECTIVES: To assess the feasibility of tedizolid, a new oxazolidinone, for the treatment of Mycobacterium avium and Mycobacterium abscessus. METHODS: We determined MICs of tedizolid for 113 isolates of NTM. Synergy with key antimycobacterial drugs was assessed using the chequerboard method and calculation of the FIC index (FICI). We performed time-kill kinetics assays of tedizolid alone and combined with amikacin for M. abscessus and with ethambutol for M. avium. Human macrophages were infected with M. abscessus and M. avium and subsequently treated with tedizolid; intracellular and extracellular cfu were quantified over time. RESULTS: NTM isolates generally had a lower MIC of tedizolid than of linezolid. FICIs were lowest between tedizolid and amikacin for M. abscessus (FICI = 0.75) and between tedizolid and ethambutol for M. avium (FICI = 0.72). Clarithromycin and tedizolid showed initial synergy, which was abrogated by erm(41)-induced macrolide resistance (FICI = 0.53). Tedizolid had a weak bacteriostatic effect on M. abscessus and combination with amikacin slightly prolonged its effect. Tedizolid had concentration-dependent activity against M. avium and its efficacy was enhanced by ethambutol. Both combinations had a concentration-dependent synergistic effect. Tedizolid could inhibit the intracellular bacterial population of both M. avium and M. abscessus. CONCLUSIONS: Tedizolid should be further investigated in pharmacodynamic studies and clinical trials for M. avium complex pulmonary disease. It is less active against M. abscessus, but still promising.


Assuntos
Infecções por Mycobacterium não Tuberculosas , Oxazolidinonas , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana , Quimioterapia Combinada , Humanos , Hansenostáticos , Macrolídeos/uso terapêutico , Testes de Sensibilidade Microbiana , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Micobactérias não Tuberculosas , Oxazolidinonas/uso terapêutico , Tetrazóis
11.
J Infect Chemother ; 26(4): 335-342, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31839561

RESUMO

BACKGROUND: Mycobacterium leprae causes leprosy and ofloxacin is used to control this bacterium. However, specific amino acid substitutions in DNA gyrases of M. leprae interferes with the effect of ofloxacin. METHODOLOGY/PRINCIPAL FINDINGS: Here we tested the inhibitory effect of WQ-3810 on DNA gyrases in M. leprae, using recombinant gyrases. We theorized that WQ-3810 and DNA gyrases interacted, which was tested in silico. Compared with control drugs like ofloxacin, WQ-3810 showed a better inhibitory effect on ofloxacin-resistant DNA gyrases. The in-silico study showed that, unlike control drugs, a specific linkage between a R1 group in WQ-3810 and aspartic acid at position 464 in the subunit B of DNA gyrases existed, which would enhance the inhibitory effect of WQ-3810. This linkage was confirmed in a further experiment, using recombinant DNA gyrases with amino acid substitutions in subunits B instead. CONCLUSIONS/SIGNIFICANCE: The inhibitory effect of WQ-3810 was likely enhanced by the specific linkage between a R1 group residue in its structure and DNA gyrases. Using interactions like the one found in the present work may help design new fluoroquinolones that contribute to halt the emergence of antibiotic-resistant pathogens.


Assuntos
Antibacterianos/farmacologia , Azetidinas/farmacologia , DNA Girase/efeitos dos fármacos , Fluoroquinolonas/farmacologia , Mycobacterium leprae/efeitos dos fármacos , Antibacterianos/uso terapêutico , Azetidinas/uso terapêutico , Farmacorresistência Bacteriana/efeitos dos fármacos , Fluoroquinolonas/uso terapêutico , Humanos , Hanseníase/tratamento farmacológico , Testes de Sensibilidade Microbiana , Ofloxacino/farmacologia
12.
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
13.
PLoS Negl Trop Dis ; 13(12): e0007946, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31881061

RESUMO

BACKGROUND: Although leprosy is efficiently treated by multidrug therapy, resistance to first-line (dapsone, rifampin) and second-line (fluoroquinolones) drugs has been described worldwide. However, the characteristics of drug resistance in Southwest China remain unknown. Furthermore, the sensitivity of polymerase chain reaction (PCR)/sequencing for resistance detection is limited, especially for paucibacillary (PB) leprosy patients. The current study aimed to develop a nested PCR/sequencing and TaqMan SNP Genotyping Assay to increase the sensitivity of the method used to detect drug resistance in Mycobacterium leprae and to reveal the nature of M. leprae drug resistance in Southwest China. METHODOLOGY/PRINCIPAL FINDINGS: Seventy-six specimens, including skin biopsy (n = 64), formalin-fixed paraffin-embedded (FFPE) (n = 11) and skin-slit smear (SSS) (n = 1) samples from multibacillary (MB, n = 70) and PB (n = 6) leprosy patients from Southwest China, were included in this study. The presence of mutations in drug resistance-determining regions (DRDRs) of the rpoB, folP1, and gyrA genes, which are associated with rifampicin, dapsone, and quinolone resistance, respectively, was detected by PCR/sequencing, as recommended by the WHO, and the nested PCR and TaqMan SNP Genotyping Assay developed in this study. Mutations in the folP gene were detected in 19 (25.00%) samples, indicating dapsone-resistant M. leprae, with one (1.31%) sample showing mutations in two genes, folP and gyrA, reflecting multidrug-resistant strains to dapsone and ofloxacin. However, no rpoB mutation was detected. Compared with PCR/sequencing, nested PCR increased the sensitivity of detecting rpoB (from 51.39% to 78.94% for leprosy patients and from 0.00% to 50.00% for PB), gyrA (from 75.00% to 80.26% for leprosy patients and from 50.00% to 66.67% for PB), and folP1 (from 5.26% to 84.21% for leprosy patients and from 0.00% to 66.67% for PB). Moreover, the TaqMan SNP Genotyping Assay showed greater sensitivity for folP1 detection (from 5.26% to 78.94-86.84% for leprosy patients and from 0.00% to 33.33%-83.33% for PB patients) than the PCR/sequencing method. In addition, the latter method was able to more easily distinguish heterozygous genotypes and mutant homozygous genotypes from homozygous genotypes. CONCLUSIONS/SIGNIFICANCE: Nested PCR/sequencing and the TaqMan SNP Genotyping Assay are rapid and highly sensitive methods for detecting drug resistance in leprosy cases. The current study revealed that diamino-diphenylsulfone (DDS; also known as dapsone) resistance in M. leprae, as indicated by folP1 gene detection, is still the most concerning form of drug resistance in leprosy patients from Southwest China.


Assuntos
Farmacorresistência Bacteriana , Técnicas de Genotipagem/métodos , Hanseníase/microbiologia , Testes de Sensibilidade Microbiana/métodos , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/genética , Reação em Cadeia da Polimerase/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , China , Feminino , Genes Bacterianos , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium leprae/isolamento & purificação , Sensibilidade e Especificidade , Análise de Sequência de DNA/métodos , Adulto Jovem
14.
Emerg Microbes Infect ; 8(1): 1479-1489, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31621517

RESUMO

Reports on antimicrobial resistance (AMR) of Mycobacterium leprae, relationship with bacteriological index (BI), and transmission in China are limited. We investigated the emergence of AMR mutations, the relationship between BI and AMR in complete, moderate and lack of BI decline cases, and molecular epidemiological features of AMR cases by enrolling 290 leprosy cases from four endemic provinces. Seven (2.41%), one (0.34%), five (1.72%), one (0.34%), and one (0.34%) strains had single mutations in folP1, rpoC, gyrA, gyrB, and 23S rRNA, respectively. Double mutations in folP1 and gyrA, rpoB and gyrA, and gyrA and 23S rRNA were observed in one (0.34%) strain each. Mutated strains occurred in three out of 81 (95% CI-0.005-0.079, p = 0.083) cases with complete BI decline, in seven out of 103 (95% CI 0.018-0.117, p = 0.008) cases with moderate BI decline, and in four out of 34 (95% CI 0.003-0.231, p = 0.044) cases with lack of BI decline. Most of these mutated strains were geographically separated and diverged genotypically. AMR mutations may not be the main cause of the lack of BI decline. The low transmission of AMR strains at the county level indicates an ongoing transmission at close contact levels.


Assuntos
Farmacorresistência Bacteriana , Hansenostáticos/farmacologia , Hanseníase/microbiologia , Mycobacterium leprae/efeitos dos fármacos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , China/epidemiologia , Feminino , Humanos , Hanseníase/epidemiologia , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mutação , Mycobacterium leprae/classificação , Mycobacterium leprae/genética , Mycobacterium leprae/isolamento & purificação , Filogenia , Adulto Jovem
15.
Tuberculosis (Edinb) ; 115: 63-66, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30948178

RESUMO

The Mycobacterium tuberculosis mec+-cysO-cysM gene cluster was shown to be part of a novel cysteine biosynthesis pathway in vitro, but little is known about its essentiality or role in M. tuberculosis physiology. In this study, we generate a knock out of the mec+-cysO-cysM gene cluster in M. tuberculosis and show that the gene cluster is not essential under a variety of conditions, suggesting redundancy in pathways for cysteine biosynthesis in M. tuberculosis. The cysteine biosynthesis gene cluster is essential for resistance for clofazimine, a peroxide-producing anti-leprosy drug. Therefore, although under most conditions the pathway is not essential, it likely has an important role in defense against oxidative stress in M. tuberculosis.


Assuntos
Antituberculosos/farmacologia , Clofazimina/farmacologia , Cisteína/biossíntese , Genes Bacterianos/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Vias Biossintéticas/genética , Cisteína/genética , Farmacorresistência Bacteriana/genética , Deleção de Genes , Hansenostáticos/farmacologia , Testes de Sensibilidade Microbiana , Família Multigênica/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Estresse Oxidativo/efeitos dos fármacos
16.
Indian J Dermatol Venereol Leprol ; 85(4): 388-392, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30950409

RESUMO

Background: Dermatophytosis is a major public health problem in our country. Although resistance to conventional oral and topical antifungal agents is being increasingly encountered, the sensitivity pattern of dermatophytes has not been systematically analysed. Aims: We aimed to determine the sensitivity pattern of dermatophyte isolates to amphotericin B and six oral antifungal drugs. Materials and Methods: Patients with dermatophytosis attending the outpatient department of dermatology were enrolled in the study. Samples were collected for mycological examination and in vitro antifungal sensitivity testing was done by broth microdilution as per the Clinical and Laboratory Standard Institute M38-A standards. Results: A total of 804 patients were enrolled. Specimens from 185 patients (23%) were both KOH and culture positive, and 44 of these isolates (41 Trichophyton mentagrophytes and 3 Trichophyton rubrum) were subjected to sensitivity testing. Minimum inhibitory concentrations (MIC) of itraconazole, ketoconazole, voriconazole and amphotericin B were comparable. The median MIC to fluconazole was higher than the other tested drugs. Dermatophytes were most susceptible to ketoconazole and voriconazole, followed by itraconazole, amphotericin B, fluconazole and griseofulvin. A high incidence of resistance was found to terbinafine and the difference was statistically significant in comparison to fluconazole, itraconazole, voriconazole, ketoconazole (P = 0.001) and griseofulvin (P = 0.003). The strains were more sensitive to amphotericin B as compared to griseofulvin (P = 0.02) and terbinafine (P < 0.001). Limitations: This was a hospital-based study and may not reflect the true pattern in the community. Only a few of the isolates were selected for study. The clinical response of patients, whose isolates were studied for in vitro sensitivity of the antifungals, was not studied. Conclusions: The sensitivity pattern of dermatophytes to various antifungals including amphotericin B, ketoconazole, voriconazole and itraconazole were determined. The studied isolates were least susceptible to terbinafine.


Assuntos
Anfotericina B/farmacologia , Antifúngicos/farmacologia , Trichophyton/efeitos dos fármacos , Administração Oral , Antifúngicos/administração & dosagem , Farmacorresistência Fúngica , Fluconazol/farmacologia , Griseofulvina/farmacologia , Humanos , Técnicas In Vitro , Índia , Itraconazol/farmacologia , Cetoconazol/farmacologia , Testes de Sensibilidade Microbiana , Terbinafina/farmacologia , Tinha/tratamento farmacológico , Tinha/microbiologia , Voriconazol/farmacologia
17.
BMC Public Health ; 19(1): 395, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30971228

RESUMO

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) outcomes are adversely impacted by delay in diagnosis and treatment. METHODS: Mixed qualitative and quantitative approaches were utilized to identify healthcare system related barriers to implementation of molecular diagnostics for MDR-TB. Randomly sampled districts from the 5 highest TB burden regions were enrolled during the 4th quarter of 2016. District TB & Leprosy Coordinators (DTLCs), and District AIDS Coordinators (DACs) were interviewed, along with staff from all laboratories within the selected districts where molecular diagnostics tests for MDR-TB were performed. Furthermore, the 2015 registers were audited for all drug-susceptible but retreatment TB cases and TB collaborative practices in HIV clinics, as these patients were in principal targeted for drug susceptibility testing by rapid molecular diagnostics. RESULTS: Twenty-eight TB districts from the 5 regions had 399 patients reviewed for retreatment with a drug-susceptible regimen. Only 160 (40%) had specimens collected for drug-susceptibility testing, and of those specimens only 120 (75%) had results communicated back to the clinic. MDR-TB was diagnosed in 16 (13.3%) of the 120 specimens but only 12 total patients were ultimately referred for treatment. Furthermore, among the HIV/AIDS clinics served in 2015, the median number of clients with TB diagnosis was 92 cases [IQR 32-157] yet only 2 people living with HIV were diagnosed with MDR-TB throughout the surveyed districts. Furthermore, the districts generated 53 front-line healthcare workers for interviews. DTLCs with intermediate or no knowledge on the clinical application of XpertMTB/RIF were 3 (11%), and 10 (39%), and DACs with intermediate or no knowledge were 0 (0%) and 2 (8%) respectively (p = 0.02). Additionally, 11 (100%) of the laboratories surveyed had only the 4-module XpertMTB/RIF equipment. The median time that XpertMTB/RIF was not functional in the 12 months prior to the investigation was 2 months (IQR 1-4). CONCLUSIONS: Underutilization of molecular diagnostics in high-risk groups was a function of a lack of front-line healthcare workforce empowerment and training, and a lack of equipment access, which likely contributed to the observed delay in MDR-TB diagnosis in Tanzania.


Assuntos
Antituberculosos/uso terapêutico , Pessoal de Saúde/psicologia , Acesso aos Serviços de Saúde/estatística & dados numéricos , Tempo para o Tratamento/estatística & dados numéricos , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Adulto , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana/estatística & dados numéricos , Pessoa de Meia-Idade , Mycobacterium tuberculosis , Patologia Molecular/estatística & dados numéricos , Poder Psicológico , Tanzânia , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico
18.
Int J Food Microbiol ; 300: 43-52, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31035250

RESUMO

The spoilage of foods caused by the growth of undesirable yeast species is a problem in the food industry. Yeast species such as Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Debaryomyces hansenii, Kluyveromyces lactis and Saccharomyces cerevisiae have been encountered in foods such as high sugar products, fruit juices, wine, mayonnaise, chocolate and soft drinks. The demand for new methods of preservations has increased because of the negative association attached to chemical preservatives. The sequence of a novel short peptide (KKFFRAWWAPRFLK-NH2) was modified to generate three versions of this original peptide. These peptides were tested for the inhibition of the yeasts mentioned above, allowing for the better understanding of their residue modifications. The range of the minimum inhibitory concentration was between 25 and 200 µg/mL. Zygosaccharomyces bailii was the most sensitive strain to the peptides, while Zygosaccharomyces rouxii was the most resistant. Membrane permeabilisation was found to be responsible for yeast inhibition at a level which was a two-fold increase of the MIC (400 µg/mL). The possibility of the production of reactive oxygen species was also assessed but was not recognised as a factor involved for the peptides' mode of action. Their stability in different environments was also tested, focusing on high salt, pH and thermal stability. The newly designed peptides showed good antifungal activity against some common food spoilage yeasts and has been proven effective in the application in Fanta Orange. These efficient novel peptides represent a new source of food preservation that can be used as an alternative for current controversial preservatives used in the food industry.


Assuntos
Microbiologia de Alimentos/métodos , Conservantes de Alimentos/farmacologia , Peptídeos/farmacologia , Leveduras/efeitos dos fármacos , Antifúngicos/farmacologia , Indústria de Processamento de Alimentos , Sucos de Frutas e Vegetais/microbiologia , Kluyveromyces/efeitos dos fármacos , Kluyveromyces/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Leveduras/crescimento & desenvolvimento , Zygosaccharomyces/crescimento & desenvolvimento
19.
mBio ; 9(5)2018 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-30301850

RESUMO

New drugs are needed to control the current tuberculosis (TB) pandemic caused by infection with Mycobacterium tuberculosis We report here on our work with AX-35, an arylvinylpiperazine amide, and four related analogs, which are potent antitubercular agents in vitro All five compounds showed good activity against M. tuberculosis in vitro and in infected THP-1 macrophages, while displaying only mild cytotoxicity. Isolation and characterization of M. tuberculosis-resistant mutants to the arylvinylpiperazine amide derivative AX-35 revealed mutations in the qcrB gene encoding a subunit of cytochrome bc 1 oxidase, one of two terminal oxidases of the electron transport chain. Cross-resistance studies, allelic exchange, transcriptomic analyses, and bioenergetic flux assays provided conclusive evidence that the cytochrome bc 1 -aa 3 is the target of AX-35, although the compound appears to interact differently with the quinol binding pocket compared to previous QcrB inhibitors. The transcriptomic and bioenergetic profiles of M. tuberculosis treated with AX-35 were similar to those generated by other cytochrome bc 1 oxidase inhibitors, including the compensatory role of the alternate terminal oxidase cytochrome bd in respiratory adaptation. In the absence of cytochrome bd oxidase, AX-35 was bactericidal against M. tuberculosis Finally, AX-35 and its analogs were active in an acute mouse model of TB infection, with two analogs displaying improved activity over the parent compound. Our findings will guide future lead optimization to produce a drug candidate for the treatment of TB and other mycobacterial diseases, including Buruli ulcer and leprosy.IMPORTANCE New drugs against Mycobacterium tuberculosis are urgently needed to deal with the current global TB pandemic. We report here on the discovery of a series of arylvinylpiperazine amides (AX-35 to AX-39) that represent a promising new family of compounds with potent in vitro and in vivo activities against M. tuberculosis AX compounds target the QcrB subunit of the cytochrome bc 1 terminal oxidase with a different mode of interaction compared to those of known QcrB inhibitors. This study provides the first multifaceted validation of QcrB inhibition by recombineering-mediated allelic exchange, gene expression profiling, and bioenergetic flux studies. It also provides further evidence for the compensatory role of cytochrome bd oxidase upon QcrB inhibition. In the absence of cytochrome bd oxidase, AX compounds are bactericidal, an encouraging property for future antimycobacterial drug development.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Mycobacterium tuberculosis/efeitos dos fármacos , Piperazinas/farmacologia , Tuberculose/tratamento farmacológico , Amidas/farmacologia , Amidas/uso terapêutico , Animais , Linhagem Celular , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Tuberculose/microbiologia
20.
FEMS Yeast Res ; 18(8)2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30137293

RESUMO

Mold and yeast contamination constitutes a major problem in food commodities, including dairy products, hence new natural preventive measures are in high demand. The aim of the current study is to identify and characterize novel antifungal peptides produced by lactic acid bacteria (LAB) in sour cream. By the use of a newly developed image-based 96-well plate fungal growth inhibition assay targeting Debaryomyces hansenii, combined with a range of analytical tools comprising HPLC-high-resolution mass spectrometry, ultrahigh-performance liquid chromatography-Triple Quadrupole MS and nuclear magnetic resonance spectroscopy, we successfully identified a new antifungal peptide (DMPIQAFLLY; 1211 Da) in sour cream enriched with two bioprotective LAB strains. This peptide represents a fragment of casein, the most abundant protein in milk. Presumably, the proteolytic activity of these bioprotective strains results in the observed 4-fold higher concentration of the peptide during storage. Both bioprotective strains are able to generate this peptide in concentrations up to 0.4 µM, independently of the sour cream starter culture employed. The peptide attenuates the growth rate of D. hansenii at concentrations ≥35 µM, and results in smaller cells and more compact colonies. Hence, the peptide is likely contributing to the overall preserving effect of the investigated bioprotective LAB strains.


Assuntos
Antifúngicos/farmacologia , Produtos Fermentados do Leite/microbiologia , Debaryomyces/efeitos dos fármacos , Lactobacillus/crescimento & desenvolvimento , Lactobacillus/metabolismo , Peptídeos/farmacologia , Antifúngicos/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Peptídeos/isolamento & purificação
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