Artykuły w czasopismach na temat „Genetic methods for resistance detection”
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SUNDSFJORD, ARNFINN, GUNNAR S. SIMONSEN, BJORG C. HALDORSEN, HAKON HAAHEIM, STIG-OVE HJELMEVOLL, PIA LITTAUER i KRISTIN H. DAHL. "Genetic methods for detection of antimicrobial resistance". APMIS 112, nr 11-12 (grudzień 2004): 815–37. http://dx.doi.org/10.1111/j.1600-0463.2004.apm11211-1208.x.
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Golovnin, A. V., A. L. Khanin i V. I. Golovnin. "THE SURGICAL STAGE OF PULMONARY TUBERCULOSIS TREATMENT USING MOLECULAR-GENETIC METHODS TO TEST SUSCEPTIBILITY TO RIFAMPICIN". Tuberculosis and Lung Diseases 97, nr 3 (3.04.2019): 31–34. http://dx.doi.org/10.21292/2075-1230-2019-97-3-31-34.
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The objective of the study: to analyze the frequency and patterns of drug resistance of Mycobacterium tuberculosis (MTB) according to the results of microbiological tests of surgical specimens of the patients who underwent surgery due to tuberculosis, and to compare them with the results of sputum tests done in the pre-operative period. Subjects and methods. The data of surgical specimens from 170 patients operated due to tuberculosis were analyzed. The surgical specimens were sent for histological and microbiological tests (detection of MTB DNA and rifampicin resistance by GeneXpert, culture on solid media with drug sensitivity testing). Results. The molecular genetic testing of surgical specimens by GeneXpert was highly effective for detection of rifampicin resistance; in 97.8% of cases, there was a match with the results of sputum culture with consecutive DST performed before the surgery. Molecular genetic tests of surgical specimens allowed detecting MTB DNA in 66.1% of patients in whom no MTB or MTB DNA was detected in sputum and bronchial washings prior to the surgery, and of them in 28.2% of cases, rifampicin resistance was detected, which was unknown before the surgery. These data allowed prescribing adequate chemotherapy immediately after surgery.
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Chroma, Magdalena, i Milan Kolar. "GENETIC METHODS FOR DETECTION OF ANTIBIOTIC RESISTANCE: FOCUS ON EXTENDED-SPECTRUM β-LACTAMASES". Biomedical Papers 154, nr 4 (1.12.2010): 289–96. http://dx.doi.org/10.5507/bp.2010.044.
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SHAMPUTA, I. C., L. RIGOUTS AND i F. PORTAELS. "Molecular genetic methods for diagnosis and antibiotic resistance detection of mycobacteria from clinical specimens". APMIS 112, nr 11-12 (grudzień 2004): 728–52. http://dx.doi.org/10.1111/j.1600-0463.2004.apm11211-1203.x.
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Eliseev, P. I., I. V. Tarasova i A. O. Mariandyshev. "MOLECULAR-GENETIC METHODS OF DETECTION OF TUBERCULOSIS AND ITS DRUG RESISTANCE IN ARKHANGELSK REGION IN 2017". Russian Journal of Infection and Immunity 8, nr 4 (16.01.2019): 567–68. http://dx.doi.org/10.15789/2220-7619-2018-4-6.14.
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Kolbert, C. P., J. Arruda, P. Varga-Delmore, X. Zheng, M. Lewis, J. Kolberg i D. H. Persing. "Branched-DNA Assay for Detection of themecA Gene in Oxacillin-Resistant and Oxacillin-Sensitive Staphylococci". Journal of Clinical Microbiology 36, nr 9 (1998): 2640–44. http://dx.doi.org/10.1128/jcm.36.9.2640-2644.1998.
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The identification of methicillin-resistant staphylococcus isolates in the clinical laboratory has typically been performed by using methods that detect phenotypic expression of resistance determinants. However, these methods may be difficult to interpret and some isolates do not express resistance until selective pressure is administered. Assays that detect genetic determinants are not subject to these limitations and have been effective in distinguishing isolates that are capable of expressing the resistance phenotype. In this study, a novel branched-DNA (bDNA) hybridization assay was used to test for themecA gene in 416 clinical staphylococcal isolates. The results were compared with those obtained by a PCR-based assay and oxacillin disk diffusion. For 155 Staphylococcus aureus and 261 coagulase-negative Staphylococcus isolates, the bDNA assay and PCR results were 100% concordant. Among the S. aureus isolates, 20 were MecA+ and 135 were MecA−. For the coagulase-negative staphylococci, 150 were MecA+ and 111 were MecA−. The results from the genotypic detection methods were compared with those obtained by oxacillin disk diffusion. No discrepancies were detected among theS. aureus isolates; however, 10 coagulase-negative isolates were MecA+ but oxacillin sensitive and 1 isolate was MecA− but oxacillin resistant. Oxacillin resistance was induced in 6 of the 10 MecA+ isolates previously classified as oxacillin sensitive. These results suggest that the bDNA method described here is a sensitive and efficient method for detection of methicillin resistance in staphylococci and that genetic detection methods may be useful for detection of potential methicillin resistance in the clinical laboratory.
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BEECH, R. N., P. SKUCE, D. J. BARTLEY, R. J. MARTIN, R. K. PRICHARD i J. S. GILLEARD. "Anthelmintic resistance: markers for resistance, or susceptibility?" Parasitology 138, nr 2 (9.09.2010): 160–74. http://dx.doi.org/10.1017/s0031182010001198.
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SUMMARYThe Consortium for Anthelmintic Resistance and Susceptibility (CARS) brings together researchers worldwide, with a focus of advancing knowledge of resistance and providing information on detection methods and treatment strategies. Advances in this field suggest mechanisms and features of resistance that are shared among different classes of anthelmintic. Benzimidazole resistance is characterized by specific amino acid substitutions in beta-tubulin. If present, these substitutions increase in frequency upon drug treatment and lead to treatment failure. In the laboratory, sequence substitutions in ion-channels can contribute to macrocyclic lactone resistance, but there is little evidence that they are significant in the field. Changes in gene expression are associated with resistance to several different classes of anthelmintic. Increased P-glycoprotein expression may prevent drug access to its site of action. Decreased expression of ion-channel subunits and the loss of specific receptors may remove the drug target. Tools for the identification and genetic analysis of parasitic nematodes and a new online database will help to coordinate research efforts in this area. Resistance may result from a loss of sensitivity as well as the appearance of resistance. A focus on the presence of anthelmintic susceptibility may be as important as the detection of resistance.
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Tsarev, V. N., E. V. Ippolitov i E. N. Nikolaeva. "PREVALENCE OF GENETIC MARKERS OF RESISTANCE TO ANTIBIOTICS IN BIOFILM-FORMING STRAINS OF OBLIGATE AND ELECTIVE ANAEROBES". Journal of microbiology epidemiology immunobiology, nr 2 (28.04.2017): 74–80. http://dx.doi.org/10.36233/0372-9311-2017-2-74-80.
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Aim. Comparative study of frequency of detection of genetic markers of resistance to antibiotics forming in anaerobic bacteria under the conditions of mixed biofilms in a clinical setting and comparison of data of phenotypic and genotypic methods of study. Materials and methods. 66 strains of bacteria forming biofilm with PCR detection of antibiotics were studied: Streptococcus sanguinis, Streptococcus salivarius, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa and anaerobic pathogens - Porphyromonas gingivalis, Tannerella forsythia, Parvinonas micra, Prevotella intermedia. Modelling of microbial biofilms in vitro and scanning electron microscopy were carried out. Results. The studied strains of resident and pathogenic microbiota were established to have genes that code resistance to P-lactam antibiotics, carbapenems, macrolides, tetracyclines. Genetic markers of resistance to p-lactam antibiotics (STX-M и МЕСА - cepha-losporines), including carbapenems (VIM and NDM, but not Oxa-48), glycopeptides (VanA and VanB), macrolides (ERM), tetracycline (Tet) and QNRB plasmids (fluoroquinolones) were detected in strains by PCR. Conclusion. The most frequently used preparations in dental practice - metronidazole and lincomycin (for the last 20 - 30 years) have shown the highest number of resistant strains - 52.3 and 22.7%, respectively. The frequency of detection of genetic markers of resistance to other studied preparations did not exceed 2.5 - 11.4%. Minimal quantity of resistant strains of anaerobic bacteria was detected for carbapenems and fluoroquinolones.
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Bhattacharyya, Roby, Jamin Liu, Peijun Ma, Nirmalya Bandyopadhyay, Jonathan Livny i Deborah Hung. "Rapid Phenotypic Antibiotic Susceptibility Testing Through RNA Detection". Open Forum Infectious Diseases 4, suppl_1 (2017): S33. http://dx.doi.org/10.1093/ofid/ofx162.082.
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Abstract Background Culture-based antibiotic susceptibility testing, the gold standard, is too slow to guide early antibiotic selection, while newer genotypic methods require comprehensive knowledge of resistance mechanisms to predict phenotype. Quantitative measurement of key antibiotic-responsive transcripts offers a rapid, phenotypic assay for assessing antibiotic susceptibility, agnostic to the genetic basis for resistance. Methods We performed RNA-Seq on Klebsiella pneumoniae and Acinetobacter baumanii treated with ciprofloxacin, gentamicin, or meropenem for 0, 10, 30, and 60 minutes. For each, we identified 50 responsive transcripts whose expression levels differ most between susceptible and resistant organisms upon antibiotic exposure. We measured their expression using a multiplexed fluorescent RNA hybridization assay (NanoString) in 69 clinical isolates, including a “test set” of multidrug-resistant strains from the CDC, in an 8-hour assay. Gene expression data from test strains were compared against known susceptible and resistant isolates to generate a transcriptional susceptibility metric. We also designed NanoString probes to detect 5 carbapenemase genes (KPC-2, KPC-3, NDM-1, OXA-48, and CTX-M15). Results Across all bacteria-antibiotic pairs tested, a susceptibility metric derived from these transcriptional assays correctly grouped isolates in 167 of 173 tests (Table 1), with only 1 of 88 resistant isolates misclassified as susceptible. Five of six incorrectly grouped isolates were within one dilution of the breakpoint MIC, including the misclassified resistant isolate. Conclusion We demonstrate phenotypic antibiotic resistance detection based on fluorescent RNA detection in an 8-hour assay. We have previously published proof-of-concept studies that this assay may be run on a positive blood culture bottle with minimal sample processing. By coupling this phenotypic assay with detection of genetic resistance determinants (demonstrated for carbapenemases) in a single assay, strains with unexplained resistance can be prioritized for further study. Disclosures All authors: No reported disclosures.
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Gupta, Gunjan, Vibhor Tak i Purva Mathur. "Detection of AmpC β Lactamases in Gram-negative Bacteria". Journal of Laboratory Physicians 6, nr 01 (styczeń 2014): 001–6. http://dx.doi.org/10.4103/0974-2727.129082.
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ABSTRACT AmpC β-lactamases are clinically important cephalosporinases encoded on the chromosomes of many Enterobacteriaceae and a few other organisms, where they mediate resistance to cephalothin, cefazolin, cefoxitin, most penicillins, and β-lactamase inhibitor/β-lactam combinations. The increase in antibiotic resistance among Gram-negative bacteria is a notable example of how bacteria can procure, maintain and express new genetic information that can confer resistance to one or several antibiotics. Detection of organisms producing these enzymes can be difficult, because their presence does not always produce a resistant phenotype on conventional disc diffusion or automated susceptibility testing methods. These enzymes are often associated with potentially fatal laboratory reports of false susceptibility to β-lactams phenotypically. With the world-wide increase in the occurrence, types and rate of dissemination of these enzymes, their early detection is critical. AmpC β-lactamases show tremendous variation in geographic distribution. Thus, their accurate detection and characterization are important from epidemiological, clinical, laboratory, and infection control point of view. This document describes the methods for detection for AmpC β-lactamases, which can be adopted by routine diagnostic laboratories.
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M. Ali Basha, Nabila, i Ahmad M. Abdul kader. "Genetically Modified Crops, Production, Detection Methods and its Biosafety Implications: A Scientific Review". Arab Journal for Plant Protection 40, nr 3 (2022): 260–79. http://dx.doi.org/10.22268/ajpp-40.3.260279.
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Ali Basha, N.M. and A.M. Abdul Kader. 2022. Genetically Modified Crops, Production, Detection Methods and its Biosafety Implications: A Scientific Review. Arab Journal of Plant Protection, 40(3): 260-279. https://doi.org/10.22268/AJPP-40.3.260279 Ensuring food security and nutrition is critical for all countries in order to overcome the problems of hunger and malnutrition, taking into consideration the various current challenges of high population rate, social and political turmoil, and degradation of natural resources, forced migration and human disease pandemics. Agricultural biotechnology contributes in enhancing agricultural productivity, food security, and livelihoods. 25 years passed since the world introduced and embraced biotech crops in 1996. Such improved genetically modified (GM) crop varieties have many useful traits such as insect resistance, herbicide tolerance, resistance to biotic and abiotic stresses, in addition to improved nutrion value, and by adopting stringent science- scrutiny and safety measures. In this context, the Biosafety Cartagena Protocol was approved to ensure the safe handling, transfer and use of living organisms that have been modified using modern biotechnology. Socioeconomic and environmental benefits have been documented by credible and independent agencies around the world. Therefore, products derived from agricultural biotechnology, especially those used to manage agricultural pests, have become one of the world fastest growing agricultural trade commodities, providing food, feed, clothing, and eco-friendly biofuels. Not to mention the development of the genome editing technology using CRISPR/Cas9, which is another step closer to developing and cultivating new varieties of agricultural crops through the use of accurate, efficient and affordable techniques for genome editing. On the other hand, researchers have developed rapid and standardized methods for the detection of genetically modified plants and seeds to facilitate testing and monitoring genetic modification taking place at the global level in order to comply with the biosafety regulations and laws. Keywords: Biosafety, biosafety clearing-house (BCH), Cartagena protocol on biosafety, genetic engineering, genetically modified crops, GMCs, GMOs
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Johansson, Markus H. K., Valeria Bortolaia, Supathep Tansirichaiya, Frank M. Aarestrup, Adam P. Roberts i Thomas N. Petersen. "Detection of mobile genetic elements associated with antibiotic resistance in Salmonella enterica using a newly developed web tool: MobileElementFinder". Journal of Antimicrobial Chemotherapy 76, nr 1 (3.10.2020): 101–9. http://dx.doi.org/10.1093/jac/dkaa390.
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Abstract Objectives Antimicrobial resistance (AMR) in clinically relevant bacteria is a growing threat to public health globally. In these bacteria, antimicrobial resistance genes are often associated with mobile genetic elements (MGEs), which promote their mobility, enabling them to rapidly spread throughout a bacterial community. Methods The tool MobileElementFinder was developed to enable rapid detection of MGEs and their genetic context in assembled sequence data. MGEs are detected based on sequence similarity to a database of 4452 known elements augmented with annotation of resistance genes, virulence factors and detection of plasmids. Results MobileElementFinder was applied to analyse the mobilome of 1725 sequenced Salmonella enterica isolates of animal origin from Denmark, Germany and the USA. We found that the MGEs were seemingly conserved according to multilocus ST and not restricted to either the host or the country of origin. Moreover, we identified putative translocatable units for specific aminoglycoside, sulphonamide and tetracycline genes. Several putative composite transposons were predicted that could mobilize, among others, AMR, metal resistance and phosphodiesterase genes associated with macrophage survivability. This is, to our knowledge, the first time the phosphodiesterase-like pdeL has been found to be potentially mobilized into S. enterica. Conclusions MobileElementFinder is a powerful tool to study the epidemiology of MGEs in a large number of genome sequences and to determine the potential for genomic plasticity of bacteria. This web service provides a convenient method of detecting MGEs in assembled sequence data. MobileElementFinder can be accessed at https://cge.cbs.dtu.dk/services/MobileElementFinder/.
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Gergova, Raina T., Virna-Maria S. Tsitou i Ivan G. Mitov. "Molecular-genetic Method for Fast Direct Detection of Staphylococcus Aureus and Methicillin Resistance in Blood Cultures and Punctures". Folia Medica 61, nr 4 (31.12.2019): 559–65. http://dx.doi.org/10.3897/folmed.61.e47941.
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Background: Invasive infections caused by methicillin resistant Staphylococcus aureus and coagulase-negative staphylococci (MRSA/MRSCoN) require fast, adequate treatment. The aim of this study was to develop a faster protocol for direct detection of MRSA/MRSCoN in blood cultures and in abscess punctures based on mecA and species specific identification of S. aureus by polymerase-chain reaction (PCR). Materials and methods: We examined 77 growth-positive BACTEC blood cultures and 50 abscess punctures by routine microbiological assay and simultaneous PCR detection of MRSA/MRSCoN. The specificity of the PCR was evaluated by using DNA from another 15 microbial species for negative controls. We determined the minimum inhibitory concentration (MIC) of oxacillin, vancomycin, tigecycline, linezolid, levofloxacin, clindamycin, and erythromycin against the S. aureus isolates using the E-test. Results: In the blood cultures, the two methods detected 39.3% of MRSA, and 93.9% of MRCoNS. In the punctures, the PCR assay identified 20.9% of MRSA and 79.2% of MSSA. In the puncture cases, there were three PCR MRSA positive and culture negative samples. Screening for susceptibility to 14 antimicrobial agents demonstrated significantly higher (p<0.05) methicillin resistance in blood culture isolates than in the puncture ones (39.3% and 20.0%, respectively). Conclusion: The new PCR protocol was very fast and specific. It was more sensitive in detecting MRSA from abscess punctures than the routine microbiological techniques. This protocol will speed up the right choice of empirical therapy, which is extremely important for saving patients’ lives.
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Sękowska, Alicja, Tomasz Bogiel i Agnieszka Kaczmarek. "Evaluation of the usefulness of selected methods for the detection of carbapenemases in Klebsiella strains". Journal of Medical Microbiology 69, nr 6 (1.06.2020): 792–96. http://dx.doi.org/10.1099/jmm.0.001202.
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Introduction. Klebsiella rods, belonging to the family Enterobacteriaceae , are generally opportunistic pathogens commonly associated with nosocomial infections, especially in intensive care units. Interestingly, strains of this genus also show multi-drug resistance. In recent years, multiple studies have indicated that the prevalence of carbapenem resistance has increased rapidly among Klebsiella representatives. Aim. The aim of this study was to assess the usefulness of selected phenotypic and genotypic methods for the detection of the most important carbapenemases in Klebsiella strains. Methodology. The study involved 51 Klebsiella strains. The ability to produce carbapenemases was determined by phenotypic methods (double disc synergy test, test with four discs and three inhibitors, CarbaNP test, culture on chromogenic medium, panels of automatic method – Phoenix, CIM test and modified Hodge test). The potential for carbapenemase synthesis was also evaluated using real-time PCR, detecting bla VIM/IMP, bla KPC, bla NDM and bla OXA-48 genes. Results. Using the phenotypic methods, positive results were obtained for all of the analysed strains. Using PCR, carbapenemase synthesis potential was confirmed on the molecular level; the bla VIM gene was detected in 23 strains, the bla NDM gene in 26 strains and the bla OXA-48 gene in two strains. Conclusion. There was complete agreement between the carbapenemases detected by the genetic method and the results obtained with phenotypic methods.
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Ostankova, Y. V., A. V. Semenov, E. B. Zueva i A. A. Totolian. "IDENTIFICATION AND MOLECULAR-GENETIC CHARACTERISTICS OF THE HEPATITIS B VIRUS AMONG HIV-INFECTED PATIENTS IN ARKHANGELSK". Problems of Virology, Russian journal 64, nr 3 (20.06.2019): 105–11. http://dx.doi.org/10.18821/0507-4088-2019-64-3-105-111.
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Aim. To estimate the prevalence and characterize the hepatitis B virus among HIV-infected patients with virological failure of antiretroviral therapy in Arkhangelsk. Material and methods. HBV markers determinations (HBsAg, anti-HBs IgG, anti-HBcor IgG, DNA HBV) were performed in isolates from blood plasma samples 64 HIV-infected patients with virological failure of antiretroviral therapy (viral load >50 IU / ml after 6 months of antiretroviral therapy or an increase in viral load after primary suppression of viral replication). For the detection of the hepatitis B virus, nucleic acids were isolated using the commercial kit «AmplePrime Ribo-prep». The virus presence analysis was performing by the polymerase chain reaction (PCR) method with hybridization-fluorescence detection in “real time” using the commercial set of «AmpliSens® HBV-FL». In the future, we used the method developed by the Saint-Petersburg Pasteur Institute, which allows detecting HBV in biological material with a low viral load. Results. HBsAg-negative (occult) HBV was detect in 28 (43.8%) HIV-infected patients. Only HBV genotype D was detected, and the HBV subgenotype D1 prevailed (39.3%) compared with the HBV subgenotype D2 (32.1%) and D3 (28.6%). Serological markers in 42.8% of patients with HBV DNA were founding. Two HBV isolates with drug resistance mutations in the polymerase gene, leaded to amino acid substitutions (L180M, M204V) associated with the resistance development to lamivudine, entecavir, telbivudine and tenofovir were identifying. Conclusion. The occult (HBsAg-negative) HBV high prevalence among HIV-infected patients suggests the need to use molecular-biological diagnostic methods to identify HBV, as well as to analyze the HBV drug resistance mutation before starting antiretroviral therapy for HIV.
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Bernatzky, Robert, i David L. Mulcahy. "Marker-aided selection in a backcross breeding program for resistance to chestnut blight in the American chestnut". Canadian Journal of Forest Research 22, nr 7 (1.07.1992): 1031–35. http://dx.doi.org/10.1139/x92-137.
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Marker-assisted selection in backcross breeding is discussed in general and with specific reference to obtaining resistance to chestnut blight in American chestnut (Castaneadentata (Marsh.) Borkh.). Resistance from Chinese chestnut (Castaneamollissima Blume) is thought to be controlled by two unlinked codominant genes. Hybrids and backcross generations are available that would allow (i) the identification of DNA markers that linked to the resistance and (ii) the development of a genetic linkage map for the genome. These markers are codominant. Markers that are linked to the resistance genes would permit detection of the different genetic classes of resistance that may have similar phenotypes. Linked markers also provide the ability to identify individuals with a maximum amount of recombination surrounding the resistance loci thereby eliminating unwanted linked donor genetic material. Having molecular markers scattered throughout the genome would allow general selection against the background donor genome. This can reduce the number of backcross generations required to obtain individuals with the phenotype of C. dentata but with the blight resistance of C. mollissima. Methods are presented for the development of a set of random genomic markers.
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Varghese, Anna M., Juber Patel, Yelena Y. Janjigian, Fanli Meng, S. Duygu Selcuklu, Gopakumar Iyer, Brian Houck-Loomis i in. "Noninvasive Detection of Polyclonal Acquired Resistance to FGFR Inhibition in Patients With Cholangiocarcinoma Harboring FGFR2 Alterations". JCO Precision Oncology, nr 5 (styczeń 2021): 44–50. http://dx.doi.org/10.1200/po.20.00178.
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PURPOSE Fibroblast growth factor receptor (FGFR) 2 alterations, present in 5%-15% of intrahepatic cholangiocarcinomas (IHC), are targets of FGFR-directed therapies. Acquired resistance is common among patients who respond. Biopsies at the time of acquired resistance to targeted agents may not always be feasible and may not capture the genetic heterogeneity that could exist within a patient. We studied circulating tumor DNA (ctDNA) as a less invasive means of potentially identifying genomic mechanisms of resistance to FGFR-targeted therapies. MATERIALS AND METHODS Serial blood samples were collected from eight patients with FGFR-altered cholangiocarcinoma for ctDNA isolation and next-generation sequencing (NGS) throughout treatment and at resistance to anti-FGFR–targeted therapy. ctDNA was sequenced using a custom ultra-deep coverage NGS panel, incorporating dual index primers and unique molecular barcodes to enable high-sensitivity mutation detection. RESULTS Thirty-one acquired mutations in FGFR2, 30/31 located in the kinase domain, were identified at resistance in six of eight patients with detectable ctDNA. Up to 13 independent FGFR2 mutations were detected per patient, indicative of striking genomic concordance among resistant subclones. CONCLUSION ctDNA could be an effective means to longitudinally monitor for acquired resistance in FGFR2-altered IHC. The numerous acquired genetic alterations in FGFR2 suggest frequent polyclonal mechanisms of resistance that cannot be detected from single-site tissue biopsies.
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Varghese, Anna M., Juber Patel, Yelena Y. Janjigian, Fanli Meng, S. Duygu Selcuklu, Gopakumar Iyer, Brian Houck-Loomis i in. "Noninvasive Detection of Polyclonal Acquired Resistance to FGFR Inhibition in Patients With Cholangiocarcinoma Harboring FGFR2 Alterations". JCO Precision Oncology, nr 5 (styczeń 2021): 44–50. http://dx.doi.org/10.1200/po.20.00178.
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PURPOSE Fibroblast growth factor receptor (FGFR) 2 alterations, present in 5%-15% of intrahepatic cholangiocarcinomas (IHC), are targets of FGFR-directed therapies. Acquired resistance is common among patients who respond. Biopsies at the time of acquired resistance to targeted agents may not always be feasible and may not capture the genetic heterogeneity that could exist within a patient. We studied circulating tumor DNA (ctDNA) as a less invasive means of potentially identifying genomic mechanisms of resistance to FGFR-targeted therapies. MATERIALS AND METHODS Serial blood samples were collected from eight patients with FGFR-altered cholangiocarcinoma for ctDNA isolation and next-generation sequencing (NGS) throughout treatment and at resistance to anti-FGFR–targeted therapy. ctDNA was sequenced using a custom ultra-deep coverage NGS panel, incorporating dual index primers and unique molecular barcodes to enable high-sensitivity mutation detection. RESULTS Thirty-one acquired mutations in FGFR2, 30/31 located in the kinase domain, were identified at resistance in six of eight patients with detectable ctDNA. Up to 13 independent FGFR2 mutations were detected per patient, indicative of striking genomic concordance among resistant subclones. CONCLUSION ctDNA could be an effective means to longitudinally monitor for acquired resistance in FGFR2-altered IHC. The numerous acquired genetic alterations in FGFR2 suggest frequent polyclonal mechanisms of resistance that cannot be detected from single-site tissue biopsies.
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Karelov, A. V., N. A. Kozub, I. I. Kucheriavy, O. I. Sozinova, I. O. Sozinov, V. K. Riabchun i Ya B. Blume. "Genetic background for moderate resistance against fusarium head blight among winter wheat developed in the Forrest Steppe of Ukraine". Faktori eksperimental'noi evolucii organizmiv 26 (1.09.2020): 96–100. http://dx.doi.org/10.7124/feeo.v26.1249.
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Aim. The aim of the work was to evaluate the genetic background of resistance to Fusarium head blight in common winter wheat cultivars based on the allelic state of the TDF_076_2D gene conferring tolerance against Fusarium graminearum Schwabe and F. culmorum (W.G.Sm.) Sacc. fungi. Methods. We studied 91 winter common wheat cultivars developed in the Institute of Plant Physiology and Genetics of NAS of Ukraine. A silica-based commercial kit was used for DNA extraction. For the allelic state detection, the INDEL1 marker co-segregating with the TDF_076_2D gene was used. Results. The frequency of the resistance allele according to the marker for the gene conferring moderate resistance to the Fusarium fungi made up 0.802. Conclusions. The majority of the common wheat cultivars from the studied sample carry the resistance allele of the gene of interest. The data obtained are consistent with the results of previous research for the wider sample of the winter and spring common wheat cultivars. The cultivars with confirmed resistance allele might show lower infection level in the field and serve as a source of the gene in marker assisted selection.
Keywords: common wheat, disease resistance genes, Fusarium head blight, molecular markers.
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van der Torre, Mireille H., Lilyann Novak-Frazer i Riina Rautemaa-Richardson. "Detecting Azole-Antifungal Resistance in Aspergillus fumigatus by Pyrosequencing". Journal of Fungi 6, nr 1 (10.01.2020): 12. http://dx.doi.org/10.3390/jof6010012.
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Guidelines on the diagnosis and management of Aspergillus disease recommend a multi-test approach including CT scans, culture, fungal biomarker tests, microscopy and fungal PCR. The first-line treatment of confirmed invasive aspergillosis (IA) consists of drugs in the azole family; however, the emergence of azole-resistant isolates has negatively impacted the management of IA. Failure to detect azole-resistance dramatically increases the mortality rates of azole-treated patients. Despite drug susceptibility tests not being routinely performed currently, we suggest including resistance testing whilst diagnosing Aspergillus disease. Multiple tools, including DNA sequencing, are available to screen for drug-resistant Aspergillus in clinical samples. This is particularly beneficial as a large proportion of IA samples are culture negative, consequently impeding susceptibility testing through conventional methods. Pyrosequencing is a promising in-house DNA sequencing method that can rapidly screen for genetic hotspots associated with antifungal resistance. Pyrosequencing outperforms other susceptibility testing methods due to its fast turnaround time, accurate detection of polymorphisms within critical genes, including simultaneous detection of wild type and mutated sequences, and—most importantly—it is not limited to specific genes nor fungal species. Here we review current diagnostic methods and highlight the potential of pyrosequencing to aid in a diagnosis complete with a resistance profile to improve clinical outcomes.
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Ro, Na-Young, Raveendar Sebastin, On-Sook Hur, Gyu-Taek Cho, Bora Geum, Yong-Jik Lee i Byoung-Cheorl Kang. "Evaluation of Anthracnose Resistance in Pepper (Capsicum spp.) Genetic Resources". Horticulturae 7, nr 11 (3.11.2021): 460. http://dx.doi.org/10.3390/horticulturae7110460.
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Anthracnose (Colletotrichum spp.), is one of the major yield losing fungal disease in both pre- and post-harvest stage of pepper (Capsicum spp.) production worldwide. Among the Colletotrichum spp., C. acutatum has strong pathogenicity, which infects both immature and mature pepper fruit leads to severe economic losses in pepper production. Inheritance of anthracnose disease resistance was evaluated with 3738 pepper genetic resources which was collected from different countries and conserved at Korean genebank. The resistance analysis against pepper anthracnose (C. acutatum) was performed on detached mature green and red fruits under laboratory conditions by spray (non-wounding) and microinjection (wounding) inoculation methods. In the primary screening, about 261 accessions were appeared to be resistant against C. acutatum in spray inoculation. The resistant accessions were further evaluated with microinjection (wounding) inoculation method using the fungal (C. acutatum) isolate of pepper anthracnose. There were highly significant differences in the disease severity and distribution of disease rating scale, considering all the sources has significant genetic variation. Finally, the anthracnose resistant pepper accessions have been validated with cleaved amplified polymorphic sequence (CAPS) and high-resolution melting (HRM) markers in which, the CAPS and HRM marker analysis showed four types of genotypes such as resistant (R), susceptible (S), heterozygous (H) and Unidentified type (UT) or not detection. The Capsicum accessions showing high level of resistance to the pathogen could be used as source material in breeding programs for resistance to anthracnose disease.
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Schlum, Katrina, Kurt Lamour, Peter Tandy, Scott J. Emrich, Caroline Placidi de Bortoli, Tejas Rao, Diego M. Viteri Dillon, Angela M. Linares-Ramirez i Juan Luis Jurat-Fuentes. "Genetic Screening to Identify Candidate Resistance Alleles to Cry1F Corn in Fall Armyworm Using Targeted Sequencing". Insects 12, nr 7 (8.07.2021): 618. http://dx.doi.org/10.3390/insects12070618.
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Evolution of practical resistance is the main threat to the sustainability of transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt crops). Monitoring of resistance to Cry and Vip3A proteins produced by Bt crops is critical to mitigate the development of resistance. Currently, Cry/Vip3A resistance allele monitoring is based on bioassays with larvae from inbreeding field-collected moths. As an alternative, DNA-based monitoring tools should increase sensitivity and reduce overall costs compared to bioassay-based screening methods. Here, we evaluated targeted sequencing as a method allowing detection of known and novel candidate resistance alleles to Cry proteins. As a model, we sequenced a Cry1F receptor gene (SfABCC2) in fall armyworm (Spodoptera frugiperda) moths from Puerto Rico, a location reporting continued practical field resistance to Cry1F-producing corn. Targeted sequencing detected a previously reported Cry1F resistance allele (SfABCC2mut), in addition to a resistance allele originally described in S. frugiperda populations from Brazil. Moreover, targeted sequencing detected mutations in SfABCC2 as novel candidate resistance alleles. These results support further development of targeted sequencing for monitoring resistance to Bt crops and provide unexpected evidence for common resistance alleles in S. frugiperda from Brazil and Puerto Rico.
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Gadani, F., G. Bindler, H. Pijenburg, L. Rossi i J. Zuber. "Current PCR Methods for the Detection, Identification and Quantification of Genetically Modified Organisms(GMOs): a Brief Review". Beiträge zur Tabakforschung International/Contributions to Tobacco Research 19, nr 2 (1.07.2000): 85–96. http://dx.doi.org/10.2478/cttr-2013-0698.
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AbstractAnalytical methods based on the polymerase chain reaction (PCR) technology are increasingly used for the detection of deoxyribonucleic acid (DNA) sequences associated with genetically modified organisms (GMOs). In the European Union and Switzerland, mandatory labeling of novel foods and food ingredients consisting of, or containing GMOs is required according to food regulations and is triggered by the presence of newly introduced foreign DNA sequences, or newly expressed proteins. In order to meet regulatory and consumer demand, numerous PCR-based methods have been developed which can detect, identify and quantify GMOs in agricultural crops, food and feed. Moreover, the determination of genetic identity allows for segregation and traceability (identity preservation) throughout the supply chain of GM crops that have been enhanced with value-added quality traits. Prerequisites for GMO detection include a minimum amount of the target gene and prior knowledge of the type of genetic modification, such as virus or insect resistance traits, including controlling elements (promoters and terminators). Moreover, DNA extraction and purification is a critical step for the preparation of PCR-quality samples, particularly for processed agricultural crops such as tobacco. This paper reviews the state-of-the-art of PCR-based method development for the qualitative and quantitative determination and identification of GMOs, and includes a short summary of official and validated GMO detection methods.
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Valença, Mariana Soares, Jeane Zanini da Rocha, Ivy Bastos Ramis, Lillian Lucas Carrion, Catiúcia Madruga, Maíra Bidart de Macedo, Carlos James Scaini, Andrea von Groll i Pedro Eduardo Almeida da Silva. "Improving tuberculosis control through the partnership between university and the health system". Revista da Sociedade Brasileira de Medicina Tropical 45, nr 4 (5.07.2012): 491–95. http://dx.doi.org/10.1590/s0037-86822012005000004.
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INTRODUCTION: Tuberculosis (TB) control is linked to the availability of qualified methods for microbiological diagnostics; however, microscopy with limited sensitivity is the only method available in many locations. The objective of this study was to evaluate the introduction of culture, drug susceptibility testing (DST), and genotyping in the routine of a Municipal Program of Tuberculosis Control. METHODS: Direct microscopy of sputum and culture in Ogawa-Kudoh were performed on 1,636 samples from 787 patients. DST of positive cultures was performed by resazurin microtiter assay and genotyping by mycobacterial interspersed repetitive units-variable number tandem repeat. RESULTS: A total 91 patients with TB were identified. The culture increased case detection by 32% compared with the microscopy; acquired resistance was 3.3% and the genotyping showed high genetic diversity. CONCLUSIONS: Ogawa-Kudoh contributed significantly to the increase in case detection and is suitable for implementation in poor-resource locations. The acquired resistance rate was lower than that reported in a recent Brazilian survey. The high genetic diversity is possibly related to the high TB prevalence in the population, as well as to early detection and suitable treatment of patients. The interaction between research and health care is important for reorienting the practice, transferring technology, and improving TB control.
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Zhurilo, A. A., A. I. Barbova, Yu A. Cherednik, P. S. Trofimova, S. V. Mironchenko, O. V. Pavlova, A. V. Chernov i L. M. Sladkova. "COMPARISON OF GENEXPERT MTB/RIF AND GENOTYPE SYSTEMS WITH MTBDRPLUS STRIPS FOR DETECTION OF MUTATIONS THAT ARE ASSOCIATED WITH M. TUBERCULOSIS RESISTANCE TO RIFAMPICIN IN TUBERCULOSIS". Ukrainian Pulmonology Journal 30, nr 4 (2022): 34–41. http://dx.doi.org/10.31215/2306-4927-2022-30-4-34-41.
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COMPARISON OF GENEXPERT MTB/RIF AND GENOTYPE SYSTEMS WITH MTBDRPLUS STRIPS FOR DETECTION OF MUTATIONS, ASSOCIATED WITH M. TUBERCULOSIS RESISTANCE TO RIFAMPICIN IN TUBERCULOSIS A. A. Zhurilo, A. I. Barbova, Yu. A. Cherednik, P. S. Trofi mova, S. V. Mironchenko, O. V. Pavlova, A. V. Chernov, L. M. Sladkova Summary The aim was to analyze the level of compliance of two molecular genetic methods GeneXpert MTB/RIF and GenoTypeDRplus in determining the drug resistance of M. tuberculosis to rifampicin when detecting mutations in the RRDR region associated with drug resistance. Object and methods. We studied strains of M. tuberculosis with resistance to rifampicin, which was detected by any of the studied molecular genetic methods. 96 sputum samples were taken. Sputum smears were examined for the presence of acid-fast bacteria by microscopy after staining with the Ziehl-Neelsen method. The material was inoculated into Middlebrook 7H9 broth and on Lowenstein-Jensen medium. The liquid medium was incubated in the BACTEC MGIT system. An immunochromatographic test was used to identify the strains. The drug susceptibility test of M. tuberculosis to rifampicin was performed using the BACTEC MGIT system. The GeneXpert MTB/RIF test was performed according to the manufacturer’s instructions. The GenoTypeDRplus assay was performed on decontaminated and concentrated sputum samples. The process was carried out in three stages: DNA extraction from the processed sputum sample; amplification of the RRDR region by PCR; hybridization of the PCR product to specific oligonucleotide probes immobilized on the test strip. For sequencing, M. tuberculosis DNA isolation was performed using the QIAamp® DNA Mini Kit. The DNA concentration was measured on a Denovix Quantus spectrophotometer. Targeted panel amplification was performed using the Deeplex Myc-TB kit. Amplicon purification was performed using Agencourt AMPure XP magnetic beads. Quantitative analysis of the purified amplification products was performed using a Qubit fluorometer. The M. tuberculosis DNA library was prepared for sample sequencing using the Nextera XT DNA library preparation kit. The library used 5.0 μl of input DNA at a concentration of 0.2 ng/μl. Sequencing was performed on MiSeq equipment with the library normalization and denaturation protocol. Results and discussion. The GeneXpert MTB/RIF and MTBDRplus systems target the same 81 bp rifampicin resistance domain. (RRDR) subunits of bacterial RNA polymerase (rpoB) for mutation detection using DNA probes, i.e. there is a correspondence of probes to each other and an expected similarity of probe binding. We analyzed all sputum samples using GeneXpert MTB/RIF and GenoType MTBDRplus and phenotypic BACCTEC MGIT methods. The level of agreement between two molecular genetic methods for the detection of rifampicin-associated mutations in the RRDR region has been established. The RRDR 81bp region of the rpoB gene of mismatched cases was studied by sequencing. GeneXpert MTB/RIF and GenoType DRplus matched the phenotypic method in 92.7% and 89.6% of cases of M. tuberculosis resistance, respectively. Complete agreement between the results of GeneXpert MTB/RIF and GenoTypeMTBDRplus was observed in 92.7% of cases. GeneXpert MTB/RIF and GenoType DRplus showed a similar pattern of binding failure of wild type probes (WT-probes) when scanning the 81 bp region (RRDRdomain), which leads to stability diagnostics through probe failure software. Sequencing of the RRDR region of “mismatched” strains showed that GeneXpert probes detected seven “mismatched” cases correctly, and GenoTypeDRplus was erroneous in all cases. GeneXpert has demonstrated greater accuracy in R-resistance detection for mismatched isolates compared to GenoTypeDRplus. GeneXpert MTB/RIF has a number of other benefits over GenoTypeDRplus. GeneXpert MTB/RIF is relatively easier to implement, biosafety requirements are minimal, study times are shorter, and the study process is more automated, resulting in less human error and more reproducible results. Given these facts and the results of GeneXpert MTB/RIF found in the study, it is recommended that GeneXpert MTB/RIF be used to detect MDR-TB. Conclusions. Sequencing of the 81bp RRDR region of mismatched M. tuberculosis strains showed that GeneXpert MTB/RIF performed more accurately than GenoTypeDRplus in detecting mutations associated with rifampicin resistance. GeneXpert MTB/RIF is relatively easier to perform, biosafety requirements are minimal, time to study is shorter, and the study process is more automated, resulting in less human error and greater reproducibility of results, so it is reasonable to use it for the detection of multidrug-resistant tuberculosis. The Deeplex Myc-TB analytical solution delivers a wealth of insightful information from antimycobacterial drug resistance markers and speeds up data analysis with easy-to-use software. Key words: tuberculosis, Mycobacterium tuberculosis, drug resistance, GeneXpert MTB/RIF and GenoTypeDRplus molecular genetic systems, BACTEC MGIT phenotypic system, sequencing. Ukr. Pulmonol. J
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Howard, Patricia A. "Aspirin Resistance". Annals of Pharmacotherapy 36, nr 10 (październik 2002): 1620–24. http://dx.doi.org/10.1345/aph.1c013.
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OBJECTIVE: To review the literature addressing the problem of aspirin resistance in patients with vascular disease. DATA SOURCES: A MEDLINE search (1966–February 2002) was performed. Key search terms included aspirin, resistance, resistant, failure, tolerance, and nonresponder. English-language studies were identified as well as pertinent references from these articles. DATA SYNTHESIS: Aspirin resistance has been reported in patients with cardiovascular, cerebrovascular, and peripheral vascular disease. Because of differences in the definition of resistance, variations in detection methods, and a lack of controlled trials, the true significance of the problem remains unknown. Multiple mechanisms for resistance have been proposed, including increased reactivity to platelet aggregating factors, genetic polymorphism, and alternate pathways for thromboxane synthesis. The studies to date have failed to demonstrate consistent relationships between aspirin's platelet-inhibiting effects, the impact of dosage escalation, and clinical outcomes. CONCLUSIONS: For many patients, aspirin is an effective antithrombotic agent. However, patients taking aspirin may demonstrate highly variable responses to in vitro tests for platelet aggregation and may experience breakthrough thromboembolic events. Although this phenomenon has been termed aspirin resistance, the lack of a uniform definition or agreement on diagnostic criteria precludes definitive recommendations at this time. In addition, strategies are needed to identify patients at risk for aspirin resistance who might benefit from alternative or combined antiplatelet therapy.
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JALAVA, JARI, i HARRI MARTTILA. "Application of molecular genetic methods in macrolide, lincosamide and streptogramin resistance diagnostics and in detection of drug-resistant Mycobacterium tuberculosis". APMIS 112, nr 11-12 (grudzień 2004): 838–55. http://dx.doi.org/10.1111/j.1600-0463.2004.apm11211-1209.x.
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Rosendorff, Adam, i David M. Dorfman. "Activated Protein C Resistance and Factor V Leiden: A Review". Archives of Pathology & Laboratory Medicine 131, nr 6 (1.06.2007): 866–71. http://dx.doi.org/10.5858/2007-131-866-apcraf.
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Abstract Context.—Factor V Leiden (FVL) is the most common heritable cause of venous thrombosis. It is caused by a single nucleotide substitution resulting in an R506Q missense mutation, resulting in factor V resistance to activated protein C (APC) inactivation. Carriers of FVL have an increased susceptibility to venous thrombosis, which is further increased in the presence of other genetic or environmental risk factors. Objective.—To review the biology, clinical findings, laboratory detection methods, and screening recommendations for patients with the FVL mutation. Data Sources.—PubMed review of published literature and online information. Conclusions.—FVL remains an important heritable cause of hypercoagulability since its discovery more than 10 years ago. Clinical suspicion should be high in cases of unexplained venous thrombosis. APC resistance and FVL mutation can be diagnosed with high sensitivity and specificity with use of clotting time–based functional assays and genetic assays, respectively, allowing for evidence-guided clinical decision making regarding the benefit of long-term anticoagulation.
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Klingler, Karl R., Thomas Junold i Klaus Wielckens. "Activated Protein C Resistance: Automated Detection of the Factor V Leiden Mutation by Mismatch Hybridization". Clinical Chemistry 45, nr 11 (1.11.1999): 1925–31. http://dx.doi.org/10.1093/clinchem/45.11.1925.
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Abstract Background: A single point mutation in the factor V gene has been demonstrated to be the cause of factor Va resistance to proteolytic cleavage by activated protein C. Knowledge of the patient’s genetic disposition is of great importance in situations such as pregnancy, surgery, use of oral contraceptives, and immobilization. Methods: We have developed a rapid, automated test for the detection of the factor V mutation that makes use of differences in thermal stability between perfect-match and non-perfect-match hybrids. A DNA fragment spanning the mutation is amplified with a biotin-labeled primer. Ruthenium-labeled oligonucleotides, perfectly matching either the biotinylated wild-type strand or the biotinylated mutation strand, are added. Heating to 95 °C and subsequent cooling lead to the formation of double-stranded DNA. Under the conditions chosen, ruthenium-labeled oligonucleotides form stable, double-stranded DNA with the biotinylated strand only if both strands perfectly match each other. The ruthenium signal is measured on a modified Elecsys 1010 system (Roche Diagnostics). Results: The ratio between the signals obtained with perfectly matching and non-perfectly matching oligonucleotides reflects the genetic status. Analyzed samples can be divided into three nonoverlapping groups based on these ratios. We confirmed the reliability of the method by analyzing several samples of known genetic status; the results were identical in every single instance. Conclusions: The test discriminates unambiguously between the heterozygous and the homozygous states. Because of its low costs and easy handling, the assay is suitable for use in routine laboratories of clinical chemistry.
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Snyder, Jessica L., Brendan Manning, Robert Shivers, Daniel Gamero, Heidi Giese, Nu Phung, Benjamin Chang i in. "655. Detection of Antibiotic Resistance Genes in Clinical Samples using T2 Magnetic Resonance". Open Forum Infectious Diseases 6, Supplement_2 (październik 2019): S301. http://dx.doi.org/10.1093/ofid/ofz360.723.
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Abstract Background Antibiotic-resistant bacteria are spread through selective pressure from the use of broad-spectrum empirical therapies, mobile genetic elements that pass resistance genes between species, and the inability to rapidly and appropriately respond to their presence. Resistance gene identification is often performed with post culture molecular diagnostic tests. The T2Resistance Panel, which detects methicillin resistance genes mecA/C; vancomycin resistance genes vanA/B; carbapenemases blaKPC, blaOXA-48,blaNDM, blaVIM, and blaIMP; AmpC β-lactamases blaCMY and blaDHA; and extended-spectrum β-lactamases blaCTX-M directly from patient blood samples, is based on T2 magnetic resonance (T2MR), an FDA-cleared technology with demonstrated high sensitivity and specificity for culture-independent bacterial and fungal species identification. Here we report the clinical performance of T2MR detection of resistance genes directly from patient blood samples. Methods Patients with a clinical diagnosis of sepsis and an order for blood culture (BC) were enrolled in the study at two sites. BCs were managed using standard procedures and MALDI-TOF for species identification. Resistance testing with the T2MR assay was performed on a direct patient draw and compared with diagnostic test results from concurrent BC specimen and BC specimen taken at other points in time. The potential impact on therapy was evaluated through patient chart review. Results T2MR detected the same resistance genes as detected by post culture diagnostics in 100% of samples from concurrent blood draws. Discordant results occurred when T2MR was taken ≥48 hours after BC for patients on antimicrobial therapy. The average time to positive result was 5.9 hours with T2MR vs. 30.6 hours with post-culture molecular testing. Conclusion The T2Resistance Panel detected antibiotic resistance genes in clinical samples and displayed agreement with post culture genetic testing. T2MR results were achieved faster than culture-dependent diagnostic testing results and may allow for an earlier change from empiric to directed therapy. The use of culture-independent diagnostics like T2MR could enable a quicker response to antibiotic-resistant organisms for individual patients and developing outbreaks. Disclosures All authors: No reported disclosures.
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Frickmann, Hagen, Wycliffe Omurwa Masanta i Andreas E. Zautner. "Emerging Rapid Resistance Testing Methods for Clinical Microbiology Laboratories and Their Potential Impact on Patient Management". BioMed Research International 2014 (2014): 1–19. http://dx.doi.org/10.1155/2014/375681.
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Atypical and multidrug resistance, especially ESBL and carbapenemase expressing Enterobacteriaceae, is globally spreading. Therefore, it becomes increasingly difficult to achieve therapeutic success by calculated antibiotic therapy. Consequently, rapid antibiotic resistance testing is essential. Various molecular and mass spectrometry-based approaches have been introduced in diagnostic microbiology to speed up the providing of reliable resistance data. PCR- and sequencing-based approaches are the most expensive but the most frequently applied modes of testing, suitable for the detection of resistance genes even from primary material. Next generation sequencing, based either on assessment of allelic single nucleotide polymorphisms or on the detection of nonubiquitous resistance mechanisms might allow for sequence-based bacterial resistance testing comparable to viral resistance testing on the long term. Fluorescencein situhybridization (FISH), based on specific binding of fluorescence-labeled oligonucleotide probes, provides a less expensive molecular bridging technique. It is particularly useful for detection of resistance mechanisms based on mutations in ribosomal RNA. Approaches based on MALDI-TOF-MS, alone or in combination with molecular techniques, like PCR/electrospray ionization MS or minisequencing provide the fastest resistance results from pure colonies or even primary samples with a growing number of protocols. This review details the various approaches of rapid resistance testing, their pros and cons, and their potential use for the diagnostic laboratory.
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Tian, Lixia, Yi Yao, Li Yin, Lanxiang Wang, Ze An, Lin Kang, Chenglin Ru i Jinping Li. "Direct Detection of Antibiotic Resistance in Chinese Helicobacter pylori Clinical Isolates by Sequencing-Based Approach". Journal of Healthcare Engineering 2022 (15.04.2022): 1–6. http://dx.doi.org/10.1155/2022/6436256.
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Objective. The detection of Helicobacter pylori mutations that result in antimicrobial resistance can serve as a guideline of antimicrobial therapeutics and probably prevent the failure of clinical treatments. Evaluating the potential of Sanger sequencing to identify genetically resistant determinants in Helicobacter pylori clinical isolates will be important. Methods. 180 cultured strains have been tested using agar dilution for antibiotic susceptibility. NCBI BLAST was used to perform genotypic analysis on the sequencing data. Sanger sequencing was evaluated as an alternative method to detect resistant genotypes and susceptibility. Results. By the conventional E-test, resistance to levofloxacin, amoxicillin, metronidazole, and clarithromycin was 67.3%, 15.1%, 96.4%, and 25.5%, respectively. In contrast, tetracycline had no resistance. Resistance to multiple drugs was observed in 8.12% of the strains. The genetic determinants of resistance to CLA was 23s rRNA, the determinants of resistance to amoxicillin was Pbp1, the determinants of resistance to metronidazole was rdxA, and the determinants of resistance to levofloxacin were GyrA and GyrB. However, there was no association of resistance in tetracycline. Conclusion. We found increased rates of metronidazole antibiotic resistance, highlighting the necessity for alternative therapies and periodic evaluation. Sanger sequencing has proved to be highly effective and holds the potential to be implemented in policies catering to local treatments.
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Song, Yi, Fengna Dou, Zhe Zhou, Ningmin Yang, Jing Zhong, Jie Pan, Qiqi Liu, Jianzhong Zhang i Shengqi Wang. "Microarray-Based Detection and Clinical Evaluation for Helicobacter pylori Resistance to Clarithromycin or Levofloxacin and the Genotype of CYP2C19 in 1083 Patients". BioMed Research International 2018 (10.09.2018): 1–12. http://dx.doi.org/10.1155/2018/2684836.
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Background. Helicobacter pylori (H. pylori) is one of the most frequent and persistent bacterial infections that affect nearly half of the world’s population. Antibiotic resistance is a constantly evolving process and local surveillance of antibiotic resistance is warranted to guide clinicians in their choice of therapy. The aim of this study was to establish a microarray-based detection to identify H. pylori infection, clarithromycin and levofloxacin susceptibility, and CYP2C19 genetic polymorphism and guide to potential choice of proton pump inhibitor (PPI), antibiotic administration for tailored H. pylori eradication therapy. Methods. By analyzing the sequence of human genomic CYP2C19⁎2 and CYP2C19⁎3 and mutations within the 23S rRNA and gyrA gene regions conferring clarithromycin and levofloxacin resistance, respectively, we developed a microarray for individual therapy detection of H. pylori infection. Plasmids were established as positive or limit of detection (LOD) reference materials. The specificity and sensitivity of the microarray had been performed. And a total of 1083 gastric biopsy samples were tested and the Kappa value had been calculated between the array and Sanger sequencing. We also analyzed the resistance to clarithromycin and levofloxacin in China, as well as the CYP2C19 polymorphisms. Results. The LOD of detecting H. pylori was 103 CFU/mL and human genome DNA was 2 ng/μL. The detection results of 1083 gastric biopsy samples showed that 691 (63.80%) were H. pylori positive, of which 266 (38.49%) were resistant to clarithromycin, 192 (27.79%) were resistant to levofloxacin, and 61 (8.83%) were resistant to both of them. For the type of CYP2C19 polymorphism, 412 (38.04%) were homozygous fast type (HomEM), 574 (53%) were heterozygous EM (HetEM), and 97 (8.96%) were poor metabolizer (PM). Conclusions. The proposed microarray-based detection has high specificity, sensitivity, and reproducibility for detecting the resistance of clarithromycin or levofloxacin as well as CYP2C19 polymorphism, which may help to improve the clinical eradication rate of H. pylori.
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PINTYE, Alexandra, Márk Z. NÉMETH, Orsolya MOLNÁR, Áron N. HORVÁTH, Zsolt SPITZMÜLLER, Nikoletta SZALÓKI, Károly PÁL, Kálmán Z. VÁCZY i Gábor M. KOVÁCS. "Improved DNA extraction and quantitative real-time PCR for genotyping Erysiphe necator and detecting the DMI fungicide resistance marker A495T, using single ascocarps". Phytopathologia Mediterranea 59, nr 1 (14.03.2020): 97–106. http://dx.doi.org/10.36253/phyto-11098.
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DNA extraction from minute fungal samples is challenging in all genetic studies. Identification of genetic groups and population biology mostly rely on the laborious production of single conidium isolates or on field samples, including infected plant materials. This paper reports a simple and cost-effective protocol for DNA extraction from individual chasmothecia of Erysiphe necator for subsequent applications. It is a less laborious alternative for genotyping purposes than production and analysis of single conidium isolates or analysis of infected plant material from the field. Using the protocols described here for 186 E. necator samples tested, genetic groups A and B were assigned. Based on CYP51 sequences, all the samples belonged to group B, while TUB2 sequences exhibited SNPs also diagnostic for group A. Additionally, a quantitative real-time PCR detection method of single nucleotide polymorphism in the CYP51 gene associated with DMI fungicide resistance was applied. The A495T marker, associated with DMI resistance, and here reported for the first time from Hungary, was detected by quantitative real-time PCR assays and direct sequencing of CYP51. The methods developed in this study can be applied as routine tests to monitor powdery mildew populations for fungicide resistance and other genetic characteristics.
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Avila-Campos, Mario Julio, Maria Auxiliadora Roque de Carvalho, Paula Prazeres Magalhães, Carlos Américo Veiga Damasceno, Edmar Chartone-Souza i Eduardo Osório Cisalpino. "Antimicrobial resistance and plasmid detection in strains of the Bacteroides fragilis group". Revista do Instituto de Medicina Tropical de São Paulo 35, nr 1 (luty 1993): 107–10. http://dx.doi.org/10.1590/s0036-46651993000100016.
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Resistant populations of the Bacteroides fragilis group bacteria (two reference ones and two isolated from human and Callithrix penicillata marmoset) were obtained by the gradient plate technique, to clindamycin, penicillin G, metronidazole and mercuric chloride. All the four tested strains were originaly susceptible to the four antimicrobial drugs at the breakpoint used in this study. MICs determination for the four cultures gave constant values for each antimicrobial, on the several steps by the gradient plate technique. The intestinal human B. fragilis strains showed three DNA bands, that could be representative of only two plasmids in the closed covalently circular (CCC) form with molecular weights of approximately 25 and 2.5 Md. The results do not permit an association between the presence of plasmid in the human strain with the susceptibility to the studied drugs. The four strains were ß-lactamase negative in the two methods used, and no particular chromosomal genetic resistance marker was demonstred. The resistance (MIC) observed, after contact with penicillin G and mercuric chloride, were two-fold in the four tested strains
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Nayak, Manoj K., Gregory J. Daglish, Thomas W. Phillips i Paul R. Ebert. "Resistance to the Fumigant Phosphine and Its Management in Insect Pests of Stored Products: A Global Perspective". Annual Review of Entomology 65, nr 1 (7.01.2020): 333–50. http://dx.doi.org/10.1146/annurev-ento-011019-025047.
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Development of resistance in major grain insect pest species to the key fumigant phosphine (hydrogen phosphide) across the globe has put the viability and sustainability of phosphine in jeopardy. The resistance problem has been aggravated over the past two decades, due mostly to the lack of suitable alternatives matching the major attributes of phosphine, including its low price, ease of application, proven effectiveness against a broad pest spectrum, compatibility with most storage conditions, and international acceptance as a residue-free treatment. In this review, we critically analyze the published literature in the area of phosphine resistance with special emphasis on the methods available for detection of resistance, the genetic basis of resistance development, key management strategies, and research gaps that need to be addressed.
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Davies, G., M. J. Stear i S. C. Bishop. "Quantitative trait loci associated with parasitic infection in sheep". Proceedings of the British Society of Animal Science 2005 (2005): 50. http://dx.doi.org/10.1017/s1752756200009613.
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Gastrointestinal nematodes cause major losses to the UK sheep industry. As anthelmintic resistance is becoming a widespread problem alternative control methods are now sought. Breeding for improved parasite resistance is a possible control method (Woolaston and Windon, 2001). As genetic markers are now widely available there is considerable potential for application to livestock breeding through quantitative trait loci (QTL) detection and subsequent marker-assisted selection schemes. Although much work is underway, there are few published studies that identify QTL associated with parasite resistance. Therefore this study aims to identify QTL associated with parasitic nematode infection in a population of Scottish Blackface lambs using faecal egg count and Immunoglobulin A activity.
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Su, Yanbin, Yumei Liu, Huolin Shen, Xingguo Xiao, Zhansheng Li, Zhiyuan Fang, Limei Yang, Mu Zhuang i Yangyong Zhang. "Inheritance Analysis and Quantitative Trait Loci Detection of Head Splitting Resistance in Cabbage (Brassica oleracea L. var. capitata)". HortScience 50, nr 7 (lipiec 2015): 944–51. http://dx.doi.org/10.21273/hortsci.50.7.944.
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Head splitting resistance (HSR) in cabbage is an important trait closely related to appearance, yield, storability, and mechanical harvestability. In this study, a doubled haploid (DH) population derived from a cross between head splitting-susceptible inbred cabbage line 79-156 and resistant line 96-100 was used to analyze inheritance and detect quantitative trait loci (QTLs) for HSR during 2011–12 in Beijing, China. The analysis was performed using a mixed major gene/polygene inheritance method and QTL mapping. This approach, which uncovered no cytoplasmic effect, indicated that HSR can be attributed to additive-epistatic effects of three major gene pairs combined with those of polygenes. Major gene and polygene heritabilities were estimated to be 88.03% to 88.22% and 5.65% to 7.60%, respectively. Using the DH population, a genetic map was constructed with simple sequence repeat (SSR) markers anchored on nine linkage groups spanning 906.62 cM. Eight QTLs for HSR were located on chromosomes C4, C5, C7, and C9 based on 2 years of phenotypic data using both multiple-QTL mapping and inclusive composite interval mapping. The identified QTLs collectively explained 37.6% to 46.7% of phenotypic variation. Three or four major QTLs (Hsr 4.2, 7.2, 9.3, and/or 9.1) showing a relatively larger effect were robustly detected in different years or with different mapping methods. The HSR trait was shown to have a complex genetic basis. Results from QTL mapping and classical genetic analysis were consistent. Our results provide a foundation for further research on HSR genetic regulation and molecular marker-assisted selection (MAS) for HSR in cabbage.
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Kadoić Balaško, Martina, Renata Bažok, Katarina M. Mikac, Darija Lemic i Ivana Pajač Živković. "Pest Management Challenges and Control Practices in Codling Moth: A Review". Insects 11, nr 1 (3.01.2020): 38. http://dx.doi.org/10.3390/insects11010038.
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The codling moth, Cydia pomonella L., is a serious insect pest in pome fruit production worldwide with a preference for apple. The pest is known for having developed resistance to several chemical groups of insecticides, making its control difficult. The control and management of the codling moth is often hindered by a lack of understanding about its biology and ecology, including aspects of its population genetics. This review summarizes the information about the origin and biology of the codling moth, describes the mechanisms of resistance in this pest, and provides an overview of current research of resistant pest populations and genetic research both in Europe and globally. The main focus of this review is on non-pesticide control measures and anti-resistance strategies which help to reduce the number of chemical pesticides used and their residues on food and the local environment. Regular monitoring for insecticide resistance is essential for proactive management to mitigate potential insecticide resistance. Here we describe techniques for the detection of resistant variants and possibilities for monitoring resistance populations. Also, we present our present work on developing new methods to maintain effective control using appropriate integrated resistance management (IRM) strategies for this economically important perennial pest.
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Pitt, Rachel, Michelle Jayne Cole, Helen Fifer i Neil Woodford. "Evaluation of the Mycoplasma genitalium Resistance Plus kit for the detection of M. genitalium and mutations associated with macrolide resistance". Sexually Transmitted Infections 94, nr 8 (3.11.2017): 565–67. http://dx.doi.org/10.1136/sextrans-2017-053366.
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ObjectivesTo compare performance of the ResistancePlus kit (SpeeDx, Australia) with in-house methods for the detection of Mycoplasma genitalium-specific DNA and mutations associated with resistance to macrolide antimicrobials, directly from clinical specimens.MethodsAssay specificity and sensitivity was analysed using DNA from 46 non-M. genitalium organisms and standard curve analysis, respectively. A panel of archived DNA extracted from 97 M. genitalium-positive clinical specimens, for which the macrolide susceptibility genotype had been previously determined, were tested on the assay and results compared.ResultsFinal analytical specificity was 100%. Sensitivity was detected to at least 140 genome copies/µL. The assay detected M. genitalium in 92/97 (94.9%, 95% CI 88.4% to 98.3%) previously positive specimens. The genetic macrolide susceptibility assigned was concordant with previous results in 85/92 (92.4%, 95% CI 85.0% to 96.9%) specimens or 85/97 (87.6%, 95% CI: 79.4% to 93.4%) when the false-negative specimens were included. On seven (7/92, 7.6%) occasions, resistant specimens were called susceptible. Further testing resolved discrepancies for all but five (5.2%) specimens.ConclusionsThe ResistancePlus assay generally performed well in comparison to methods currently employed at the reference laboratory. It detected a range of different mutations; however, a small number of specimens that were genotyped as macrolide resistant by Sanger sequencing were either not detected by the assay or were genotyped as susceptible. This could impact on treatment outcomes if assay results were used for patient management.
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Rao, Mohan, Fairuz A. Rashid, Surianti Shukor, Rohaidah Hashim i Norazah Ahmad. "Detection of Antimicrobial Resistance Genes Associated with Carbapenem Resistance from the Whole-Genome Sequence of Acinetobacter baumannii Isolates from Malaysia". Canadian Journal of Infectious Diseases and Medical Microbiology 2020 (2.04.2020): 1–9. http://dx.doi.org/10.1155/2020/5021064.
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Background. The spread of carbapenem-resistant A. baumannii (CrAb) is gaining worldwide attention. The spread of this pathogen is largely due to its ability to acquire various resistance genes of intrinsic and extrinsic origins that confer unpredictable susceptibility to β-lactams. The aim of this study was to analyze β-lactamase genetic compositions of CrAb in Malaysia. Methods. Whole-genome sequencing (WGS) was carried out on 13 CrAb isolates from clinical samples in Malaysia from 2011 to 2016. Results. Endotracheal aspirate was the dominant clinical sample source (n = 6), and only one isolate was obtained from wound swab. A total of 6 sequence types (STs) of the Oxford scheme were identified, including 4 reported STs and 2 novel STs. Eleven isolates were classified into clonal complex 92 (CC92/ICII), among which ST195 and ST208 were the most prevalent STs. All 13 CrAb isolates harbored multiple β-lactamase genes. blaOXA-23 (n = 13) and blaOXA-66 (n = 11) were the dominant carbapenemase gene families found in these isolates. All isolates harbor blaADC, blaOXA-51-like, and blaOXA-23-like genes. blaTEM (n = 7), blaNDM-1 (n = 3), blaCARB-8 (n = 1), and blaPER-3 (n = 1) are amongst other β-lactamase genes found in this study. ISAba1 was found upstream to blaOXA-23 (n = 13), blaOXA-66 (n = 1), and blaADC (n = 11). All blaNDM-1 isolates had ISAba125 (mobile genetic element) upstream to the genes. All isolates were positive for Tn2006/2008 and Tn2009 but were negative for Tn2007. Conclusion. Most of the isolates were grouped under the CC92 clonal complex which belongs to international clonal lineage 2. These findings predict that carriage of carbapenem-resistant genes possibly constitutes the underlying basis of high level of international clone II prevalence. Therefore, molecular surveillance and antimicrobial stewardship are essential in implementing policies to prevent and control the spread of CrAb in hospital settings.
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Ushtanit, Anastasia, Yulia Mikhailova, Alexandra Lyubimova, Marina Makarova, Svetlana Safonova, Alexey Filippov, Sergey Borisov i Danila Zimenkov. "Genetic Profile of Linezolid-Resistant M. tuberculosis Clinical Strains from Moscow". Antibiotics 10, nr 10 (13.10.2021): 1243. http://dx.doi.org/10.3390/antibiotics10101243.
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Background: Linezolid, bedaquiline, and newer fluoroquinolones are currently placed as priority Group A drugs for the treatment of drug-resistant tuberculosis. The number of reported linezolid-resistant clinical strains is still low, and the correlation of molecular determinants with phenotype is not perfect. Methods: We determined the linezolid MICs for clinical isolates from the Moscow region and identified mutations in rplC and rrl genes. Results: All 16 linezolid-resistant isolates had previously reported mutations in the rplC or rrl loci, and 13 of them bore a RplC C154R substitution. Detection of this substitution in a heteroresistant state was not successful, probably, due to the more stable DNA secondary structure of the mutated fragment, which precludes its amplification in mixes with the wild-type DNA. Strains with an rplC mutation had higher linezolid MIC compared to isolates with rrl mutations. Conclusions: Linezolid resistance mostly emerged during treatment with the latest regimen. Three primary cases with linezolid resistance question the possible transmission of totally drug-resistant tuberculosis in the Moscow region, which demands further investigation.
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Berndt, Annerose, Adriana S. Leme, Laura K. Williams, Randy Von Smith, Holly S. Savage, Timothy M. Stearns, Shirng-Wern Tsaih i in. "Comparison of unrestrained plethysmography and forced oscillation for identifying genetic variability of airway responsiveness in inbred mice". Physiological Genomics 43, nr 1 (styczeń 2011): 1–11. http://dx.doi.org/10.1152/physiolgenomics.00108.2010.
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Lung function detection in mice is currently most accurately measured by invasive techniques, which are costly, labor intensive, and terminal. This limits their use for large-scale or longitudinal studies. Noninvasive assays are often used instead, but their accuracy for measuring lung function parameters such as resistance and elastance has been questioned in studies involving small numbers of mouse strains. Here we compared parameters detected by two different methods using 29 inbred mouse strains: enhanced pause (Penh), detected by unrestrained plethysmography, and central airway resistance and lung elastance, detected by a forced oscillation technique. We further tested whether the phenotypic variations were determined by the same genomic location in genome-wide association studies using a linear mixed model algorithm. Penh, resistance, and elastance were measured in nonexposed mice or mice exposed to saline and increasing doses of aerosolized methacholine. Because Penh differed from airway resistance in several strains and because the peak genetic associations found for Penh, resistance, or elastance were located at different genomic regions, we conclude that using Penh as an indicator for lung function changes in high-throughput genetic studies (i.e., genome-wide association studies or quantitative trait locus studies) measures something fundamentally different than airway resistance and lung elastance.
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Selyanskaya, Nadejda A., Sergey O. Vodop'yanov, Violetta A. Rykova i Elena P. Sokolova. "Transmissive Antibiotic Resistance, Associated with the SXT Element, in Cholera Vibrios Isolated in the Territory of Russia". Journal of microbiology, epidemiology and immunobiology 97, nr 3 (25.06.2020): 258–64. http://dx.doi.org/10.36233/0372-9311-2020-97-3-8.
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Aim. Detection of SXT elements in cholera vibrios O1 and nonO1/nonO139 serogroups and study of the effectiveness of their conjugative transmission to Escherichia coli cells.Materials and methods. In conjugation experiments, Vibrio cholerae O1 El Tor (3) and V. cholerae nonO1/ nonO139 (3) strains were used as donors. Donor strains, recipients, and transconjugants were tested in realtime PCR for sensitivity to antibiotics and for the presence of drug resistance genes and integrase gene (int). Electrophoresis was carried out on a 0.7% agarose gel with ethidium bromide staining.Results. Resistance to chloramphenicol, trimethoprim/sulfamethoxazole, streptomycin was transmitted in conjugation experiments with a frequency of 2.1 × 10–9–7.1 × 10–9. The genes int and dfrA1 (resistance to trimethoprim/sulfamethoxazole) were found in most V. cholerae strains, and were stably transmitted to E. coli QD Rif r cells and in reverse crosses of V. cholerae O1 El Tor 5879 Nalr .Conclusion. The detection of the SXT element in V. cholerae strains and its successful horizontal transfer emphasize the need to detect such mobile genetic elements to control the spread of antibiotic resistance in V. cholerae.
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Cervantes, Jorge, Noemí Yokobori i Bo-Young Hong. "Genetic Identification and Drug-Resistance Characterization of Mycobacterium tuberculosis Using a Portable Sequencing Device. A Pilot Study". Antibiotics 9, nr 9 (27.08.2020): 548. http://dx.doi.org/10.3390/antibiotics9090548.
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Clinical management of tuberculosis (TB) in endemic areas is often challenged by a lack of resources including laboratories for Mycobacterium tuberculosis (Mtb) culture. Traditional phenotypic drug susceptibility testing for Mtb is costly and time consuming, while PCR-based methods are limited to selected target loci. We herein utilized a portable, USB-powered, long-read sequencing instrument (MinION), to investigate Mtb genomic DNA from clinical isolates to determine the presence of anti-TB drug-resistance conferring mutations. Data analysis platform EPI2ME and antibiotic-resistance analysis using the real time ARMA workflow, identified Mtb species as well as extensive resistance gene profiles. The approach was highly sensitive, being able to detect almost all described drug resistance conferring mutations based on previous whole genome sequencing analysis. Our findings are supportive of the practical use of this system as a suitable method for the detection of antimicrobial resistance genes, and effective in providing Mtb genomic information. Future improvements in the error rate through statistical analysis, drug resistance prediction algorithms and reference databases would make this a platform suited for the clinical setting. The small size, relatively inexpensive cost of the device, as well as its rapid and simple library preparation protocol and analysis, make it an attractive option for settings with limited laboratory infrastructure.
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Ammar, Ahmed M., Marwa I. Abd El-Hamid, Rania M. S. El-Malt, Doaa S. Azab, Sarah Albogami, Mohammad M. Al-Sanea, Wafaa E. Soliman, Mohammed M. Ghoneim i Mahmoud M. Bendary. "Molecular Detection of Fluoroquinolone Resistance among Multidrug-, Extensively Drug-, and Pan-Drug-Resistant Campylobacter Species in Egypt". Antibiotics 10, nr 11 (3.11.2021): 1342. http://dx.doi.org/10.3390/antibiotics10111342.
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In recent times, resistant foodborne pathogens, especially of the Campylobacter species, have created several global crises. These crises have been compounded due to the evolution of multidrug-resistant (MDR) bacterial pathogens and the emergence of extensively drug-resistant (XDR) and pan-drug-resistant (PDR) strains. Therefore, this study aimed to investigate the development of resistance and the existence of both XDR and PDR among Campylobacter isolates. Moreover, we explored the use of the polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) technique for the detection of fluoroquinolone (FQ)-resistant Campylobacter isolates. A total of 120 Campylobacter isolates were identified depending on both phenotypic and genotypic methods. Of note, cefoxitin and imipenem were the most effective drugs against the investigated Campylobacter isolates. Interestingly, the majority of our isolates (75%) were MDR. Unfortunately, both XDR and PDR isolates were detected in our study with prevalence rates of 20.8% and 4.2%, respectively. All FQ-resistant isolates with ciprofloxacin minimum inhibitory concentrations ≥4 µg/mL were confirmed by the genetic detection of gyrA chromosomal mutation via substitution of threonine at position 86 to isoleucine (Thr-86-to-Ile) using the PCR-RFLP technique. Herein, PCR-RFLP was a more practical and less expensive method used for the detection of FQ resistant isolates. In conclusion, we introduced a fast genetic method for the identification of FQ-resistant isolates to avoid treatment failure through the proper description of antimicrobials.
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Skiba-Kurek, Iwona, Paweł Nowak, Joanna Empel, Magdalena Tomczak, Joanna Klepacka, Iwona Sowa-Sierant, Iwona Żak, Bartosz Pomierny i Elżbieta Karczewska. "Evaluation of Biofilm Formation and Prevalence of Multidrug-Resistant Strains of Staphylococcus epidermidis Isolated from Neonates with Sepsis in Southern Poland". Pathogens 10, nr 7 (11.07.2021): 877. http://dx.doi.org/10.3390/pathogens10070877.
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Staphylococcus epidermidis strains play an important role in nosocomial infections, especially in the ones associated with biofilm formation on medical devices. The paper was aimed at analyzing the mechanisms of antibiotic resistance and confirming the biofilm-forming ability among S. epidermidis strains isolated from the blood of hospitalized newborns. Genetic analysis of resistance mechanism determinants included multiplex PCR detection of mecA, ermA, ermB, ermC, msrA, and mef genes. Biofilm analysis comprised phenotypic and genotypic methods including Christensen and Freeman methods and PCR detection of the icaADB gene complex. Among the tested S. epidermidis strains, 89% of the isolates were resistant to methicillin, 67%—to erythromycin, 53%—to clindamycin, 63%—to gentamicin, and 23%—to teicoplanin, while all the strains were susceptible to vancomycin and linezolid. The mecA gene was detected in 89% of the isolates, the ermC gene was the most common and present among 56% of the strains, while the msrA gene was observed in 11% isolates. Eighty-five percent of the strains were described as biofilm-positive by phenotypic methods and carried the icaADB gene cluster. Multidrug resistance and the biofilm-forming ability in most of the strains tested may contribute to antimicrobial therapy failure (p < 0.05).
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Kong, Say Li, Huipeng Li, Joyce A. Tai, Elise T. Courtois, Huay Mei Poh, Dawn Pingxi Lau, Yu Xuan Haw i in. "Concurrent Single-Cell RNA and Targeted DNA Sequencing on an Automated Platform for Comeasurement of Genomic and Transcriptomic Signatures". Clinical Chemistry 65, nr 2 (1.02.2019): 272–81. http://dx.doi.org/10.1373/clinchem.2018.295717.
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Abstract BACKGROUND The comeasurement of both genomic and transcriptomic signatures in single cells is of fundamental importance to accurately assess how the genetic information correlates with the transcriptomic phenotype. However, existing technologies have low throughput and laborious work flows. METHODS We developed a new method for concurrent sequencing of the transcriptome and targeted genomic regions (CORTAD-seq) within the same single cell on an automated microfluidic platform. The method was compatible with the downstream library preparation, allowing easy integration into existing next-generation sequencing work flows. We incorporated a single-cell bioinformatics pipeline for transcriptome and mutation analysis. RESULTS As proof of principle, we applied CORTAD-seq to lung cancer cell lines to dissect the cellular consequences of mutations that result in resistance to targeted therapy. We obtained a mean detection of 6000 expressed genes and an exonic rate of 50%. The targeted DNA-sequencing data achieved a 97.8% detection rate for mutations and allowed for the identification of copy number variations and haplotype construction. We detected expression signatures of tyrosine kinase inhibitor (TKI) resistance, epidermal growth factor receptor (EGFR) amplification, and expansion of the T790M mutation among resistant cells. We also identified characteristics for TKI resistance that were independent of EGFR T790M, indicating that other alterations are required for resistance in this context. CONCLUSIONS CORTAD-seq allows assessment of the interconnection between genetic and transcriptomic changes in single cells. It is operated on an automated, commercially available single-cell isolation platform, making its implementation straightforward.
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Oberhettinger, Philipp, Jan Zieger, Ingo Autenrieth, Matthias Marschal i Silke Peter. "Evaluation of two rapid molecular test systems to establish an algorithm for fast identification of bacterial pathogens from positive blood cultures". European Journal of Clinical Microbiology & Infectious Diseases 39, nr 6 (4.02.2020): 1147–57. http://dx.doi.org/10.1007/s10096-020-03828-5.
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Abstract Fast identification of pathogens directly from positive blood cultures is of highest importance to supply an adequate therapy of bloodstream infections (BSI). There are several platforms providing molecular-based identification, detection of antimicrobial resistance genes, or even a full antimicrobial susceptibility testing (AST). Two of such test systems allowing rapid diagnostics were assessed in this study: The Biofire FilmArray® and the Genmark ePlex®, both fully automated test system with a minimum of hands-on time. Overall 137 BSI episodes were included in our study and compared to conventional culture–based reference methods. The FilmArray® is using one catridge including a panel for the most common bacterial and fungal BSI pathogens as well as selected resistance markers. The ePlex® offers three different cartridges for detection of Gram-positives, Gram-negatives, and fungi resulting in a broader panel including also rare pathogens, putative contaminants, and more genetic resistance markers. The FilmArray® and ePlex® were evaluated for all 137 BSI episodes with FilmArray® detecting 119 and ePlex® detecting 128 of these. For targets on the respective panel of the system, the FilmArray® generated a sensitivity of 98.9% with 100% specificity on Gram-positive isolates. The ePlex® system generated a sensitivity of 94.7% and a specificity of 90.7% on Gram-positive isolates. In each case, the two systems performed with 100% sensitivity and specificity for the detection of Gram-negative specimens covered by each panel. In summary, both evaluated test systems showed a satisfying overall performance for fast pathogen identification and are beneficial tools for accelerating blood culture diagnostics of sepsis patients.
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Vyhnánek, T., i J. Bednář. "Detection of the varietal purity in sample of harvested wheat and triticale grains by prolamin marker". Plant, Soil and Environment 49, No. 3 (10.12.2011): 95–98. http://dx.doi.org/10.17221/4096-pse.
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In 1997 and 1998 we used samples of harvested grain to verify the possibility of distinguishing 14 winter wheat genotypes and six triticale genotypes and detecting the impurity on the basis of the detection of polymorphism of prolamin kernel proteins using the methods of the PAGE ISTA. On the basis of the identity index two sister prolamin lines with different percentage of participation, which was based on the weather conditions of the year of harvest, were discovered in seven wheat genotypes (Astella, Brea, Hana, Ilona, Siria, Sofia and Šárka) and two triticale genotypes (Tornádo and KM 779). A foreign genotype was detected in the Hana and Astella varieties. The identity index of the impurity to the Astella and Hana variety (i.e. ii = 0.28 and ii = 0.20, respectively) was considerably lower. In an unknown genotype (impurity) we detected the gliadin block Gld 1B3, which is the genetic marker of rye translocation T1BL.1RS, the Sr31 gene of resistance to black rust, higher cold resistance and the marker of poor baking quality (presence of secalin genes). The results proved the potential practical application of the method of electrophoretic detection of polymorphism of prolamin proteins as markers of impurities of foreign genotypes in a seed sample.