Navegando por Autor "Palomino, Juan Carlos"

Agora exibindo 1 - 14 de 14

Item
Biological cost in Mycobacterium tuberculosis with mutations in the rpsL, rrs, rpoB, and katG genes
(2012) Spies, Fernanda Sá; Groll, Andrea Von; Ribeiro, Andrezza Wolowski; Soares, Daniela Fernandes Ramos; Ribeiro, Marta Osório; Costa, Elis Regina Dalla; Martin, Anandi; Palomino, Juan Carlos; Rossetti, Maria Lucia; Zaha, Arnaldo; Silva, Pedro Eduardo Almeida da
When bacteria develop drug-resistant mutations, there is often an associated biological cost; however, some strains can exhibit low- or no-cost mutations. In the present study, a quantitative resazurin reduction assay was used to measure the biological cost of Mycobacterium tuberculosis isolates that contained different mutations in the rpsL, rrs, rpoB, and katG genes, and showed different resistance profiles. Biological costs were determined by comparing the growth curves of drug-resistant isolates with drug-susceptible strains. Some strains, such as those with rpoB mutations other than S531L and strains with mutations in all of the studied genes, grew more slowly than did drug-susceptible strains. However, some strains grew more quickly than drug-susceptible strains, such as those that had only the rpsL K43R mutation. Strains with the mutation katG S315T presented heterogeneous biological costs. When analyzed individually, strains with the mutations rpsL43/katG315, rpoB531, and rpoB531/katG315 grew faster than drug-susceptible strains. The results suggest that some strains with the most common mutations correlated to a high resistance toward streptomycin, isoniazid and rifampicin can grow as well as or better than susceptible strains.
Item
Efflux as a mechanism for drug resistance in mycobacterium tuberculosis
(2011) Silva, Pedro Eduardo Almeida da; Groll, Andrea Von; Martin, Anandi; Palomino, Juan Carlos
Tuberculosis remains an important global public health problem, with an estimated prevalence of 14 million individuals with tuberculosis worldwide in 2007. Because antibiotic treatment is one of the main tools for tuberculosis control, knowledge of Mycobacterium tuberculosis drug resistance is an important component for the disease control strategy. Although several gene mutations in specific loci of the M. tuberculosis genome have been reported as the basis for drug resistance, additional resistance mechanisms are now believed to exist. Efflux is a ubiquitous mechanism responsible for intrinsic and acquired drug resistance in prokaryotic and eukaryotic cells. Mycobacterium tuberculosis presents one of the largest numbers of putative drug efflux pumps compared with its genome size. Bioinformatics as well as direct and indirect evidence have established relationships among drug efflux with intrinsic or acquired resistance in M. tuberculosis. This minireview describes the current knowledge on drug efflux in M. tuberculosis.
Item
Evaluation of the speed-oligo mycobacteria assay for the identification of nontuberculous mycobacteria
(2015) Ramis, Ivy Bastos; Cnockaert, Margo; Groll, Andrea Von; Mathys, Vanessa; Simon, Anne; Tortoli, Enrico; Palomino, Juan Carlos; Silva, Pedro Eduardo Almeida da; Vandamme, Peter; Andre, Emmanuel; Martin, Anandi
Nontuberculous mycobacteria (NTM) causing human infectious disease have become increasingly common. Rapid and accurate identification to the species level is, therefore, critical. The Speed-Oligo Mycobacteria assay is an oligochromatographic method that was made available recently for the identification and differentiation of mycobacteria. The present study aimed to evaluate the performance of the Speed-Oligo Mycobacteria assay for the identification of NTM. We examined a total of 62 strains (9 type strains, 19 reference strains and 34 clinical isolates) belonging to 13 different species (Mycobacterium intracellulare, M. fortuitum, M. gordonae, M. kansasii, M. marinum, M. peregrinum, M. scrofulaceum, M. abscessus, M. bovis BCG, M. chelonae, M. avium, M. malmoense and M. xenopi). The Speed-Oligo Mycobacteria assay was performed according to the manufacturer’s instructions. Discrepant results between Speed-Oligo Mycobacteria and the original identification were reassessed by the Speed-Oligo Mycobacteria assay and resolved by the GenoType Mycobacterium CM assay and by sequencing of 16S rRNA and protein-encoding genes. We found 93.5 % (58/62) concordance for the identification of NTM as compared with the original identification. Three strains were erroneously identified by Speed-Oligo Mycobacteria: one M. kansasii strain was identified as Mycobacterium tuberculosis complex, and one M. chelonae strain and one M. peregrinum strain were both identified as Mycobacterium abscessus. Moreover, one M. chelonae strain was not identified by Speed-Oligo Mycobacteria since it did not react with any species-specific probe. For these strains, sequencing of the genes hsp65, 16S rRNA and rpoB and the GenoType Mycobacterium CM assay were performed. The Speed-Oligo Mycobacteria assay can be a useful tool for the rapid and easy identification of the most common NTM. If applied in clinical practice it could reduce diagnostic delays and contribute to correct clinical and better management of infections caused by NTM.
Item
Fitness of Mycobacterium tuberculosis strains of the W-Beijing and Non-W-Beijing genotype
(2010) Groll, Andrea Von; Martin, Anandi; Stehr, Matthias; Singh, Mahavir; Portaels, Françoise; Silva, Pedro Eduardo Almeida da; Palomino, Juan Carlos
Background: Multidrug resistant tuberculosis (MDR-TB) is a major threat for global tuberculosis control. The W-Beijing Mycobacterium tuberculosis genotype has been associated with drug resistance. Elucidation of the mechanisms underlying this epidemiological finding may have an important role in the control of MDR-TB. The aim of this study was to evaluate the fitness of drug-susceptible and MDR M. tuberculosis strains of the W-Beijing genotype compared with that of Non-W-Beijing strains. Methodology/Principal Findings: Fitness of M. tuberculosis strains was determined by evaluating the difference in the growth curves obtained in the MGIT960 automated system and assessing the competitive growth capacity between WBeijing and non-W-Beijing strains. The W-Beijing MDR strains had a significant longer lag phase duration compared to the other strains but did not present a significant fitness cost. When grown in competition they had an advantage only in medium containing 0.1% Tween 80. Conclusions/Significance: It was not possible to confirm a selective advantage of W-Beijing strains to grow, except for differences in their resistance to Tween 80. Further studies are needed to elucidate the putative advantage of W-Beijing strains compared to other genotypes.
Item
Fitness study of the RDRio lineage and LAM family of mycobacterium tuberculosis in the city of Rio Grande, Brazil
(2010) Groll, Andrea Von; Martin, Anandi; Felix, Carolina Rodrigues; Prata, Pedro Fernandes Sanmartin; Honscha, Gunther; Portaels, Françoise; Vandamme, Peter; Silva, Pedro Eduardo Almeida da; Palomino, Juan Carlos
RDRio is a novel Mycobacterium tuberculosis lineage of the Latin American– Mediterranean (LAM) family. LAM has been found worldwide but is more predominant in South America. The aim of this study was to assess the presence of the RDRio lineage and LAM family in the city of Rio Grande, Brazil, and to investigate the fitness of these strains based on determination of their growth rate. Fifty clinical isolates of M. tuberculosis were genotyped and 43 different patterns were found by spoligotyping and mycobacterial interspersed repetitive units–variable number of tandem repeats. The predominant genotypes belonged to the LAM family (54% of the strains) followed by clade T (22%) and Haarlem (16%). The RDRio lineage represented 38% of the total strains and 70.4% of the LAM strains found in this study. Strains belonging to the LAM family showed a fitness advantage when comparing their rate of growth with that of non-LAM strains, but a significant difference between RDRio and non-RDRio strains was not confirmed.
Item
Fluoroquinolone resistance in mycobacterium tuberculosis and mutations in gyra and gyrb
(2009) Groll, Andrea Von; Martin, Anandi; Jureen, Pontus; Hoffner, Sven; Vandamme, Peter; Portaels, Françoise; Palomino, Juan Carlos; Silva, Pedro Eduardo Almeida da
This study evaluated cross-resistance of Mycobacterium tuberculosis strains to ofloxacin, moxifloxacin, and gatifloxacin and investigated the presence of mutations in gyrA and gyrB. Fluoroquinolone susceptibilities were determined for 41 M. tuberculosis strains by the proportion method on 7H11, and MICs were determined by the resazurin microtiter assay. Forty strains shared the same resistance results for the three fluoroquinolones. However, one strain, with an Asn-533 3 Thr mutation in gyrB, was susceptible to ofloxacin but resistant to moxifloxacin and gatifloxacin.
Item
Fluoroquinolone resistance in mycobacterium tuberculosis and mutations in gyra and gyrb
(2009) Groll, Andrea Von; Martin, Anandi; Jureen, Pontus; Hoffner, Sven; Vandamme, Peter; Portaels, Françoise; Palomino, Juan Carlos; Silva, Pedro Almeida da
This study evaluated cross-resistance of Mycobacterium tuberculosis strains to oﬂoxacin, moxiﬂoxacin, and gatiﬂoxacin and investigated the presence of mutations in gyrA and gyrB. Fluoroquinolone susceptibilities were determined for 41 M. tuberculosis strains by the proportion method on 7H11, and MICs were determined by the resazurin microtiter assay. Forty strains shared the same resistance results for the three ﬂuoroquinolones.However, one strain, with an Asn-533 3 Thr mutation in gyrB, was susceptible to oﬂoxacin but resistant to moxiﬂoxacin and gatiﬂoxacin.
Item
Growth kinetics of mycobacterium tuberculosis measured by quantitative resazurin reduction assay: a tool for fitness studies
(2010) Groll, Andrea Von; Martin, Anandi; Portaels, Françoise; Silva, Pedro Eduardo Almeida da; Palomino, Juan Carlos
We standardized a method to evaluate the growth kinetics of Mycobacterium tuberculosis by measuring quantitatively the reduction of resazurin by spectrophotometry. Growth curves and the rate of growth of twenty-one M. tuberculosis clinical isolates were determined. The method showed technical simplicity and is inexpensive to assess the fitness of each isolate.
Item
Molecular basis and mechanisms of drug resistance in Mycobacterium tuberculosis: classical and new drugs
(2011) Silva, Pedro Eduardo Almeida da; Palomino, Juan Carlos
Tuberculosis (TB) remains one of the leading public health problems worldwide. Declared as a global emergency in 1993 by the WHO, its control is hampered by the emergence of multidrug resistance (MDR), defined as resistance to at least rifampicin and isoniazid, two key drugs in the treatment of the disease. More recently, severe forms of drug resistance such as extensively drug-resistant (XDR) TB have been described. After the discovery of several drugs with anti-TB activity, multidrug therapy became fundamental for control of the disease. Major advances in molecular biology and the availability of new information generated after sequencing the genome of Mycobacterium tuberculosis increased our knowledge of the mechanisms of resistance to the main anti-TB drugs. Better knowledge of the mechanisms of drug resistance in TB and the molecular mechanisms involved will help us to improve current techniques for rapid detection and will also stimulate the exploration of new targets for drug activity and drug development. This article presents an updated review of the mechanisms and molecular basis of drug resistance in M. tuberculosis. It also comments on the several gaps in our current knowledge of the molecular mechanisms of drug resistance to the main classical and new anti-TB drugs and briefly discusses some implications of the development of drug resistance and fitness, transmission and pathogenicity of M. tuberculosis.
Item
Nitrate reductase assay for the rapid detection of pyrazinamide resistance in Mycobacterium tuberculosis using nicotinamide
(2008) Martin, Anandi; Cubillos-Ruiz, Andrés; Groll, Andrea Von; Portillo, Patricia Del; Portaels, Françoise; Palomino, Juan Carlos
Objectives: The purpose of this study was to develop the nitrate reductase assay (NRA) for the rapid detection of pyrazinamide resistance in Mycobacterium tuberculosis using nicotinamide resistance as a marker of pyrazinamide resistance in Lo¨wenstein–Jensen (LJ) medium at neutral pH. Methods: We tested 68 M. tuberculosis isolates using nicotinamide at three different concentrations (1000, 500 and 250 mg/L) by the NRA in LJ medium and compared the results with those obtained with the BACTEC 460-TB or the BACTEC MGIT 960 as reference standard methods. Mutations in the pncA gene were detected by DNA sequencing of the pyrazinamide-resistant isolates. Results: Out of 34 M. tuberculosis pyrazinamide-resistant isolates, 31 were found to be resistant to 1000 and 500 mg/L nicotinamide giving sensitivity and specificity of 91% and 94%, respectively. At 250 mg/L nicotinamide, the sensitivity and specificity decreased to 91% and 71%, respectively. Results were obtained in an average of 10 days. Based on these results, a tentative breakpoint concentration of 500 mg/L nicotinamide was defined. DNA sequencing of the pncA gene detected mutations in 26 out of 34 M. tuberculosis isolates resistant to pyrazinamide. Conclusions: The NRA using nicotinamide to detect resistance to pyrazinamide in LJ medium is a rapid and accurate method that could be useful in limited-resource countries where the BACTEC 460-TB or the BACTEC MGIT 960 system is not available.
Item
Nitrate reductase assay using sodium nitrate for rapid detection of multidrug resistant tuberculosis
(2012) Macedo, Maíra Bidart de; Groll, Andrea Von; Fissette, Krista; Palomino, Juan Carlos; Silva, Pedro Eduardo Almeida da; Martin, Anandi
We validated the nitrate reductase assay (NRA) for the detection of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) using sodium nitrate (NaNO3) in replacement of potassium nitrate (KNO3) as nitrate source. NaNO3 is cheaper than KNO3 and has no restriction on use which facilitates the implementation of NRA to detect MDR-TB.
Item
Rapid detection of mycobacterium tuberculosis resistance to second-line drugs by use of the manual mycobacterium growth indicator tube system
(2008) Martin, Anandi; Groll, Andrea Von; Fissette, Krista; Palomino, Juan Carlos; Varaine, Francis; Portaels, Françoise
The objective of this study was to evaluate the manual mycobacterium growth indicator tube (MGIT) system for the testing of Mycobacterium tuberculosis susceptibility to second-line drugs compared to the proportion method. One hundred eighty-eight M. tuberculosis isolates were tested for susceptibility to oﬂoxacin, kanamycin, ethionamide, and capreomycin by the manual MGIT, and results were compared to those obtained with the proportion method on 7H11 agar, considered a reference method. Results for oﬂoxacin and capreomycin were excellent, with 100% accuracy, and a result of 99.4% accuracy was achieved for kanamycin. For ethionamide,accuracy was lower, with a result of 86.7% compared to that of the proportion method. We proposed the following critical concentrations for the drugs: for oﬂoxacin, 2.0 g/ml; for kanamycin, 2.5 g/ml; for ethionamide, 5 g/ml; and for capreomycin, 2.5 g/ml. The time required to obtain results was an average of 8 days by the manual MGIT and 3 weeks by the reference method. Our results show that the manual MGIT is an accurate method for the rapid susceptibility testing of M. tuberculosis to second-line drugs. There is no need for a machine when using the manual MGIT, and results can be read with a simple UV lamp or with a semiquantitative reader, which considerably reduces the cost of the method.
Item
Rifampin-isoniazid oligonucleotide typing: an alternative format for rapid detection of multidrug-resistant mycobacterium tuberculosis
(2010) Neuta, Ivan Hernandez; Varela, Andres; Martin, Anandi; Groll, Andrea Von; Jureen, Pontus; Lopez, Beatriz; Imperiale, Belen; kenders, Girts; Ritacco, Viviana; Hoffner, Sven; Morcillo, Nora; Palomino, Juan Carlos; Portillo, Patricia Del
A reverse line blot DNA hybridization format for rapid detection of multidrug-resistant tuberculosis was developed. Simultaneous detection of rifampin and isoniazid resistance in clinical isolates of Mycobacterium tuberculosis was based on the same ampliﬁcation/reverse hybridization principle of the widely used spoligotyping. The test involved probing nine DNA regions that are targets of common drug resistance-associated mutations in the genes rpoB, katG, and inhA. Addition of quaternary amine tetramethyl ammonium chloride to the hybridization buffer promoted multiple hybrid formations at a single annealing temperature irrespective of the different GC contents of probes. The assay was standardized using 20 well-documented strains from the Institute of Tropical Medicine (Belgium) and evaluated blindly in a central laboratory with 100 DNA samples that were obtained from cultured clinical isolates and shipped dried from three other countries. Compared with drug susceptibility testing, both sensitivity and speciﬁcity for rifampin resistance detection were 93.0% while for isoniazid the values were 87.7% and 97.7%, respectively. Compared with sequencing and GenoType MTBDRplus methods, sensitivity and speciﬁcity reached 96.4% and 95.5% for rifampin and 92.7% and 100% for isoniazid. Altogether, 40/45 (89%) multidrug-resistant isolates were correctly identiﬁed. Advantages of this in-house development include versatility, capacity to run up to 41 samples by triplicate in a single run, and reuse of the membrane at least 10 times. These features substantially reduce cost per reaction and make the assay an attractive tool for use in reference laboratories of countries that have a high burden of multidrugresistant tuberculosis but that cannot afford expensive commercial tests because of limited resources.
Item
Streptomycin resistance and lineage specific polymorphisms in Mycobacterium tuberculosis gidB gene
(2011) Spies, Fernanda Sá; Ribeiro, Andrezza Wolowski; Soares, Daniela Fernandes Ramos; Ribeiro, Marta Osório; Martin, Anandi; Palomino, Juan Carlos; Rossetti, Maria Lucia Rosa; Silva, Pedro Eduardo Almeida da; Zaha, Arnaldo
Mutations related to streptomycin resistance in the rpsL and rrs genes are well known and can explain about 70% of this phenotypic resistance. Recently, the gidB gene was found to be associated with low-level streptomycin resistance in Mycobacterium tuberculosis. Mutations in gidB have been reported with high frequency, and this gene appears to be very polymorphic, with frameshift and point mutations occurring in streptomycinsusceptible and streptomycin-resistant strains. In this study, mutations in gidB appeared in 27% of streptomycin- resistant strains that contained no mutations in the rpsL or rrs genes, and they were associated with low-level streptomycin resistance. However, the association of certain mutations in gidB with streptomycin resistance needs to be further investigated, as we also found mutations in gidB in streptomycin-susceptible strains. This occurred only when the strain was resistant to rifampin and isoniazid. Two specific mutations appeared very frequently in this and other studies of streptomycin-susceptible and -resistant strains; these mutations were not considered related to streptomycin resistance, but as a polymorphism. We stratified the strains according to the different phylogenetic lineages and showed that the gidB16 polymorphism (16G allele) was exclusively present in the Latin American-Mediterranean (LAM) genotype, while the gidB92 polymorphism(92C allele) was associated with the Beijing lineage in another population. In the sample studied, the two characterized single-nucleotide polymorphisms could distinguish LAM and Beijing lineages from the other lineages.