scholarly journals Evaluation of a Loop-Mediated Isothermal Amplification Assay to Detect Carbapenemases Directly From Bronchoalveolar Lavage Fluid Spiked With Acinetobacter spp.

2021 ◽  
Vol 11 ◽  
Author(s):  
Javier Moreno-Morales ◽  
Andrea Vergara ◽  
Tomislav Kostyanev ◽  
Jesús Rodriguez-Baño ◽  
Herman Goossens ◽  
...  
Keyword(s):  
Bronchoalveolar Lavage ◽  
Molecular Diagnostics ◽  
Bronchoalveolar Lavage Fluid ◽  
Lavage Fluid ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Carbapenem Resistant ◽  
Amplification Assay ◽  
Acinetobacter Spp ◽  
Selection Of

Carbapenem-resistant Acinetobacter spp. mainly Acinetobacter baumannii are frequently causing nosocomial infections with high mortality. In this study, the efficacy of the Eazyplex® SuperBug Complete A system, based on loop-mediated isothermal amplification (LAMP), to detect the presence of carbapenemases in Acinetobacter spp. directly from bronchoalveolar lavage (BAL) samples was assessed. A total of 22 Acinetobacter spp. strains producing OXA-23, OXA-40, OXA-58, NDM, and IMP were selected. Eazyplex SuperBug Complete A kit, used with the Genie II device, is a molecular diagnostics kit that detects a selection of genes that express carbapenemases (blaKPC, blaNDM, blaVIM, blaOXA–48, blaOXA–23, blaOXA–40, and blaOXA–58). Negative BAL samples were identified, McFarland solutions were prepared from each of the 22 Acinetobacter strains and serial dilutions in saline solution were made to finally spike BAL samples to a concentration of 102 and 103 CFU/ml. Fifteen concentrations out of the 44 tested out did not provide detection of the carbapenemase-producing gene, all but one being at the lowest concentration tested at 102 CFU/ml; therefore, the limit of sensitivity is 103 CFU/ml. This assay represents the kind of advantages that investing in molecular diagnostics brings to the clinical practice, allowing the identification of carbapenemases in less than 30 min with a sensitivity of 103 CFU/ml.

scholarly journals Evaluation of a Loop-Mediated Isothermal Amplification assay to detect carbapenemases from Acinetobacter spp. directly from bronchoalveolar lavage fluid

2020 ◽  
Author(s):  
J. Moreno-Morales ◽  
A. Vergara ◽  
T. Kostyanev ◽  
J. Rodriguez-Baño ◽  
H. Goossens ◽  
...  
Keyword(s):  
Bronchoalveolar Lavage ◽  
Acinetobacter Baumannii ◽  
Nosocomial Infections ◽  
Bronchoalveolar Lavage Fluid ◽  
Lavage Fluid ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Carbapenem Resistant ◽  
Amplification Assay ◽  
Acinetobacter Spp

AbstractCarbapenem-resistant Acinetobacter spp. mainly Acinetobacter baumannii are frequently causing nosocomial infections with high mortality. In this study, the efficacy of the Eazyplex® SuperBug Complete A system (Amplex Diagnostics GmbH, Gars-Bahnhof, Germany), based on loop-mediated isothermal amplification (LAMP), to detect the presence of carbapenemases in Acinetobacter spp. directly from bronchoalveolar lavage samples was assessed, detecting all tested carbapenemases in less than 30 minutes with a sensitivity of 103 CFU/ml.

scholarly journals Development of a Rapid Loop-Mediated Isothermal Amplification Assay for the Detection of Dothistroma septosporum

Forests ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 362
Author(s):  
Colin L. Myrholm ◽  
Bradley D. Tomm ◽  
Renate Heinzelmann ◽  
Nicolas Feau ◽  
Richard C. Hamelin ◽  
...  
Keyword(s):  
Lamp Assay ◽  
Isothermal Amplification ◽  
Pine Needle ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Assay ◽  
Lecanosticta Acicola ◽  
Dothistroma Pini ◽  
Biology Laboratory ◽  
Dothistroma Septosporum ◽  
Selection Of

A Loop-Mediated Isothermal Amplification (LAMP) assay was developed for the detection of the pine pathogen Dothistroma septosporum (G. Dorog.) M. Morelet. The specificity of the LAMP assay was tested using a selection of pine needle fungi, including Dothistroma pini Hulbary, and Lecanosticta acicola (Thüm.) Syd.; only D. septosporum DNA was amplified by the test. In terms of sensitivity, the assay was able to detect as little as 1 pg of total D. septosporum DNA. This assay enables DNA extracted from diseased host needles to be rapidly tested for the presence of D. septosporum using relatively simple to operate equipment away from a fully equipped molecular biology laboratory.

scholarly journals Pathogenic characteristics of sputum and bronchoalveolar lavage fluid samples from patients with lower respiratory tract infection

2020 ◽  
Author(s):  
Zheng Peng ◽  
Jin’an Zhou ◽  
lei tian
Keyword(s):  
Klebsiella Pneumoniae ◽  
Respiratory Tract ◽  
Bronchoalveolar Lavage ◽  
Lower Respiratory Tract ◽  
Detection Rate ◽  
Quality Evaluation ◽  
Bronchoalveolar Lavage Fluid ◽  
Lavage Fluid ◽  
Antimicrobial Sensitivity ◽  
Carbapenem Resistant

Abstract Background: Lower respiratory tract infections（LRIs）were a very common disease, no matter in community acquired infection or hospital acquired infection. Sputum and bronchoalveolar lavage fluid (BALF) were the most important specimens of LRIs. The choice of antibiotics for the treatment of LRIs usually depended on the results of antimicrobial sensitivity of bacteria isolated from sputum and BALF. However, it was rarely reported to compare the pathogens isolated from sputum and BALF and the difference of antimicrobial sensitivity.Methods: A retrospective study was conducted to analyze the differences between sputum and BALF samples in pathogen isolation and antimicrobial sensitivity in our hospital. Results: In our hospital during 2013-2015 year, the quality evaluation of sputum samples was not conducted before sputum culture, but in 2016-2018 year, the quality evaluation of sputum samples was conducted firstly and only qualified sputum samples were cultured. The results of pathogen culture showed that Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus and Haemophilus influenzae were the top five pathogens isolated from sputum and BALF. Antimicrobial susceptibility test showed for these five pathogens the susceptibility rates of BALF isolates to most antibiotics were higher than those isolated from sputum. The sensitivity of A. baumannii to common antibiotics was less than 50%. In particular, the detection rate of carbapenem resistant A. baumannii (CR-ABA) in sputum and BALF was higher than 80%. The sensitivity of P. aeruginosa to antibiotics other than ticarcillin/clavulanic acid and minocycline were higher than 50%. The detection rate of carbapenem resistant Klebsiella pneumoniae (CR-KPN) was 10% - 20% in 2013-2015 and 30% - 50% in 2016-2018. The detection rate of MRSA in sputum was higher than 80%, while that in BALF was 65% - 70%. The separation rate of beta-lactamase-negative ampicillin-resistant H. influenzae (BLNAR) was between 0-6%.Conclusions: The sensitivity of strains isolated from BALF to commonly used antibiotics was generally higher than that from sputum. MRSA, CR-KPN and A. baumannii were the focus of infection control for LRIs.

scholarly journals Progress in loop-mediated isothermal amplification assay for detection of Schistosoma mansoni DNA: towards a ready-to-use test

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
J. García-Bernalt Diego ◽  
P. Fernández-Soto ◽  
B. Crego-Vicente ◽  
S. Alonso-Castrillejo ◽  
B. Febrer-Sendra ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Molecular Diagnostics ◽  
Neglected Tropical Diseases ◽  
Point Of Care ◽  
Isothermal Amplification ◽  
Diagnostic Tools ◽  
Important Species ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Assay ◽  
Diagnostic Applications

Abstract Schistosomiasis is one of the most prevalent Neglected Tropical Disease, affecting approximately 250 million people worldwide. Schistosoma mansoni is the most important species causing human intestinal schistosomiasis. Despite significant efforts in recent decades, the global disease burden of schistosomiasis remains extremely high. This could partly be attributed to the absence of accurate diagnostic tools, primarily in endemic areas. Loop-mediated isothermal amplification (LAMP) is increasingly used in molecular diagnostics as a field-friendly alternative to many other complex molecular methods and it has been proposed as an ideal candidate for revolutionizing point-of-care molecular diagnostics. In a previous work, a LAMP-based method to detect S. mansoni DNA (SmMIT-LAMP) was developed by our research group for early diagnosis of active schistosomiasis in an experimental infection murine model. The SmMIT-LAMP has been further successfully evaluated in both human stool and snail samples and, recently, in human urine samples. In this study, we developed an important improvement for SmMIT-LAMP molecular assay, transforming it into a cold maintenance dry format suitable for potentially manufacturing as kit for ready-to-use for schistosomiasis diagnosis. This procedure could be applied to create dry LAMP kits for a laboratory setting and for diagnostic applications for other neglected tropical diseases.

Performance of a loop-mediated isothermal amplification assay (Isoplex CRE-ART) to detect common carbapenemase-encoding genes in Gram-negative bacteria

2021 ◽  
Vol 70 (7) ◽  
Author(s):  
Laura Berneking ◽  
Lucia Asar ◽  
Anna Both ◽  
Benjamin Berinson ◽  
Martin Aepfelbacher ◽  
...  
Keyword(s):  
Isothermal Amplification ◽  
Gram Negative Bacteria ◽  
Pcr Assay ◽  
Gram Negative ◽  
Content Type ◽  
Loop Mediated Isothermal Amplification ◽  
Carbapenem Resistant ◽  
Amplification Assay ◽  
Pathogen Characterization ◽  
Encoding Genes

Carbapenem-resistant Gram-negative bacteria (CR-GNB) are a major source of nosocomial infections worldwide. In this study, the ability of a loop-mediated isothermal amplification (LAMP)-based method (Isoplex CRE-ART) to rapidly detect carbapenemase-encoding genes bla OXA-48-like, bla OXA-23-like, bla OXA-24-like, bla KPC, bla VIM, bla NDM and bla IMP in 231 carbapenem-resistant Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii isolates was investigated. The accuracy of the LAMP test was compared to results of molecular isolate characterization using a Laboratory Developed Test multiplex carbapenemase PCR assay. The LAMP test correctly identified the presence of on-panel carbapenemases with a sensitivity of 99.16 % [95 % confidence interval (CI): 95.39–99.96 %] and a specificity of 98.21 % (95 % CI: 93.72–99.68 %) in 60 min. Our findings suggest that the Isoplex CRE-ART assay is able to rapidly identify carbapenemase genes in CR-GNB and improves options for pathogen characterization in the context of clinical microbiological and infection control diagnostics.

scholarly journals Antibacterial effect of acetic acid during an outbreak of carbapenem-resistant Acinetobacter baumannii in an ICU (II)

2021 ◽  
Vol 15 (08) ◽  
pp. 1167-1172
Author(s):  
Carolina Garciglia-Mercado ◽  
Ramon Gaxiola-Robles ◽  
Felipe Ascencio ◽  
Concepción Grajales-Muñiz ◽  
Maria Luisa Soriano Rodríguez ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Acinetobacter Baumannii ◽  
Environmental Samples ◽  
Antibacterial Effect ◽  
Companion Paper ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Carbapenem Resistant ◽  
Before And After ◽  
Amplification Assay

Introduction: Acetic acid (AA) has been commonly used in medicine as an antiseptic agent for the past 6000 years. This study evaluated the antibacterial effect of AA during an outbreak in an intensive care unit (ICU) facility in Baja California Sur, México. Methodology: Thirty-five environmental samples were collected, subsequently, disinfection with AA (4%) was performed, and two days later the same areas were sampled inside the ICU facility. Carbapenem-resistant A. baumannii (CRAB) was detected with loop-mediated isothermal amplification assay (Garciglia-Mercado et al. companion paper), targeting blaOXA-23-like, blaOXA-24-like, blaOXA-51-like, blaOXA-58-like, blaIMP and blaVIM genes. CRAB isolates before and after disinfection were compared by PFGE. Results: Eighteen (54.5%) and five (14.3%) of thirty-five environmental samples were identified as Acinetobacter baumannii before and after disinfection, respectively, showing a significant decrease of 85.7% (p < 0.05) both by Loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). Furthermore, the presence of blaOXA-23-like and blaOXA-58-like genes significantly decreased (p < 0.05) both by LAMP and PCR methods. PFGE genotype showed high similarity among CRAB isolates before and after disinfection, suggesting wide clonal dissemination in the ICU facility. Conclusions: This study demonstrated the novel application of AA with the LAMP assays developed for detecting CRAB. AA promises to be a cheap and efficacious disinfectant alternative to both developed and especially developing countries, preventing the spread of this organism in the environment and to other susceptible patients in health care settings.

scholarly journals MorphoCatcher: a multiple-alignment based web tool for target selection and designing taxon-specific primers in the loop-mediated isothermal amplification method

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6801
Author(s):  
Fedor V. Shirshikov ◽  
Yuri A. Pekov ◽  
Konstantin A. Miroshnikov
Keyword(s):  
Molecular Diagnostics ◽  
Target Selection ◽  
Primer Design ◽  
Multiple Alignment ◽  
Isothermal Amplification ◽  
Specific Primer ◽  
Bioinformatics Tool ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Method ◽  
Selection Of

Background Advantages of loop-mediated isothermal amplification in molecular diagnostics allow to consider the method as a promising technology of nucleic acid detection in agriculture and medicine. A bioinformatics tool that provides rapid screening and selection of target nucleotide sequences with subsequent taxon-specific primer design toward polymorphic orthologous genes, not only unique or conserved common regions of genome, would contribute to the development of more specific and sensitive diagnostic assays. However, considering features of the original software for primer selection, also known as the PrimerExplorer (Eiken Chemical Co. LTD, Tokyo, Japan), the taxon-specific primer design using multiple sequence alignments of orthologs or even viral genomes with conservative architecture is still complicated. Findings Here, MorphoCatcher is introduced as a fast and simple web plugin for PrimerExplorer with a clear interface. It enables an execution of multiple-alignment based search of taxon-specific mutations, visual screening and selection of target sequences, and easy-to-start specific primer design using the PrimerExplorer software. The combination of MorphoCatcher and PrimerExplorer allows to perform processing of the multiple alignments of orthologs for informative sliding-window plot analysis, which is used to identify the sequence regions with a high density of taxon-specific mutations and cover them by the primer ends for better specificity of amplification. Conclusions We hope that this new bioinformatics tool developed for target selection and taxon-specific primer design, called the MorphoCatcher, will gain more popularity of the loop-mediated isothermal amplification method for molecular diagnostics community. MorphoCatcher is a simple web plugin tool for the PrimerExplorer software which is freely available only for non-commercial and academic users at http://morphocatcher.ru.

scholarly journals Pathogenic Characteristics of Sputum and Bronchoalveolar Lavage Fluid Samples from Patients with Lower Respiratory Tract Infection

2020 ◽  
Author(s):  
zheng peng ◽  
jin'an zhou ◽  
lei tian
Keyword(s):  
Klebsiella Pneumoniae ◽  
Respiratory Tract ◽  
Bronchoalveolar Lavage ◽  
Lower Respiratory Tract ◽  
Detection Rate ◽  
Quality Evaluation ◽  
Bronchoalveolar Lavage Fluid ◽  
Lavage Fluid ◽  
Antimicrobial Sensitivity ◽  
Carbapenem Resistant

Abstract Background Lower respiratory tract infections(LRIs)were a very common disease, no matter in community acquired infection or hospital acquired infection. Sputum and bronchoalveolar lavage fluid (BALF) were the most important specimens of LRIs. The choice of antibiotics for the treatment of LRIs usually depended on the results of antimicrobial sensitivity of bacteria isolated from sputum and BALF. However, it was rarely reported to compare the pathogens isolated from sputum and BALF and the difference of antimicrobial sensitivity.Methods A retrospective study was conducted to analyze the differences between sputum and BALF samples in pathogen isolation and antimicrobial sensitivity in our hospital.Results In our hospital during 2013–2015 year, the quality evaluation of sputum samples was not conducted before sputum culture, but in 2016–2018 year, the quality evaluation of sputum samples was conducted firstly and only qualified sputum samples were cultured. The results of pathogen culture showed that Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus and Haemophilus influenzae were the top five pathogens isolated from sputum and BALF. Antimicrobial susceptibility test showed for these five pathogens the susceptibility rates of BALF isolates to most antibiotics were higher than those isolated from sputum. The sensitivity of A. baumannii to common antibiotics was less than 50%. In particular, the detection rate of carbapenem resistant A. baumannii (CR-ABA) in sputum and BALF was higher than 80%. The sensitivity of P. aeruginosa to antibiotics other than ticarcillin/clavulanic acid and minocycline were higher than 50%. The detection rate of carbapenem resistant Klebsiella pneumoniae (CR-KPN) was 10% − 20% in 2013–2015 and 30% − 50% in 2016–2018. The detection rate of MRSA in sputum was higher than 80%, while that in BALF was 65% − 70%. The separation rate of beta-lactamase-negative ampicillin-resistant H. influenzae (BLNAR) was between 0–6%.Conclusions The sensitivity of strains isolated from BALF to commonly used antibiotics was generally higher than that from sputum. MRSA, CR-KPN and A. baumannii were the focus of infection control for LRIs.

scholarly journals A comparative study between real-time PCR and loop-mediated isothermal amplification to detect carbapenemase and/or ESBL genes in Enterobacteriaceae directly from bronchoalveolar lavage fluid samples

2020 ◽  
Author(s):  
A Vergara ◽  
J Moreno-Morales ◽  
I Roca ◽  
C Pitart ◽  
T Kostyanev ◽  
...  
Keyword(s):  
Bronchoalveolar Lavage ◽  
Real Time ◽  
Real Time Pcr ◽  
False Negative ◽  
Carbapenem Resistance ◽  
Lavage Fluid ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Negative Results ◽  
False Negative Results

Abstract Objectives To evaluate and compare the efficacy of real-time PCR (Xpert Carba-R) and loop-mediated isothermal amplification (Eazyplex® SuperBug CRE) for detecting carbapenemase carriage in Enterobacteriaceae directly from bronchoalveolar lavage (BAL). Methods Negative BAL samples were spiked with 21 well-characterized carbapenemase-producing Enterobacteriaceae strains to a final concentration of 102–104 cfu/mL. Xpert Carba-R (Cepheid, Sunnyvale, CA, USA), which detects five targets (blaKPC, blaNDM, blaVIM, blaOXA-48 and blaIMP-1), and the Eazyplex® SuperBug CRE system (Amplex-Diagnostics GmbH, Germany), which detects seven genes (blaKPC, blaNDM, blaVIM, blaOXA-48, blaOXA-181, blaCTXM-1 and blaCTXM-9), were evaluated for the detection of these genes directly from BAL samples. Results Xpert Carba-R showed 100% agreement with carbapenemase characterization by PCR and sequencing for all final bacteria concentrations. Eazyplex® SuperBug CRE showed 100%, 80% and 27% agreement with PCR and sequencing when testing 104, 103 and 102 cfu/mL, respectively. False negative results for Eazyplex® SuperBug CRE matched the highest cycle threshold values for Xpert Carba-R. Hands-on time for both assays was about 15 min, but Eazyplex® SuperBug CRE results were available within 30 min, whereas Xpert Carba-R took around 50 min. Conclusions We here describe the successful use of two commercial diagnostic tests, Xpert Carba-R and Eazyplex® SuperBug CRE, to detect bacterial carbapenem resistance genes directly in lower respiratory tract samples. Our results could be used as proof-of-concept data for validation of these tests for this indication.

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