Multiplexed Signal Ion Emission Reactive Release Amplification (SIERRA) Assay for the Culture-Free Detection of Gram-Negative and Gram-Positive Bacteria and Antimicrobial Resistance Genes. - Related Documents




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222401.0000Multiplexed Signal Ion Emission Reactive Release Amplification (SIERRA) Assay for the Culture-Free Detection of Gram-Negative and Gram-Positive Bacteria and Antimicrobial Resistance Genes. The global prevalence of antibiotic-resistant bacteria has increased the risk of dangerous infections, requiring rapid diagnosis and treatment. The standard method for diagnosis of bacterial infections remains dependent on slow culture-based methods, carried out in central laboratories, not easily extensible to rapid identification of organisms, and thus not optimal for timely treatments at the point-of-care (POC). Here, we demonstrate rapid detection of bacteria by combining electrochemical immunoassays (EC-IA) for pathogen identification with confirmatory quantitative mass spectral immunoassays (MS-IA) based on signal ion emission reactive release amplification (SIERRA) nanoparticles with unique mass labels. This diagnostic method uses compatible reagents for all involved assays and standard fluidics for automatic sample preparation at POC. EC-IA, based on alkaline phosphatase-conjugated pathogen-specific antibodies, quantified down to 10(4) bacteria per sample when testing Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa lysates. EC-IA quantitation was also obtained for wound samples. The MS-IA using nanoparticles against S. aureus, E. coli, Klebsiella pneumoniae, and P. aeruginosa allowed selective quantitation of ∼10(5) bacteria per sample. This method preserves bacterial cells allowing extraction and amplification of 16S ribosomal RNA genes and antibiotic resistance genes, as was demonstrated through identification and quantitation of two strains of E. coli, resistant and nonresistant due to β-lactamase cefotaximase genes. Finally, the combined immunoassays were compared against culture using remnant deidentified patient urine samples. The sensitivities for these immunoassays were 83, 95, and 92% for the prediction of S. aureus, P. aeruginosa, and E. coli or K. pneumoniae positive culture, respectively, while specificities were 85, 92, and 97%. The diagnostic platform presented here with fluidics and combined immunoassays allows for pathogen isolation within 5 min and identification in as little as 15 min to 1 h, to help guide the decision for additional testing, optimally only on positive samples, such as multiplexed or resistance gene assays (6 h).202133819029
222810.9996Accurate Detection of the Four Most Prevalent Carbapenemases in E. coli and K. pneumoniae by High-Resolution Mass Spectrometry. BACKGROUND: At present, phenotypic growth inhibition techniques are used in routine diagnostic microbiology to determine antimicrobial resistance of bacteria. Molecular techniques such as PCR are often used for confirmation but are indirect as they detect particular resistance genes. A direct technique would be able to detect the proteins of the resistance mechanism itself. In the present study targeted high resolution mass spectrometry assay was developed for the simultaneous detection of KPC, OXA-48-like, NDM, and VIM carbapenemases. METHODS: Carbapenemase specific target peptides were defined by comparing available sequences in GenBank. Selected peptide sequences were validated using 62 Klebsiella pneumoniae and Escherichia coli isolates containing: 16 KPC, 21 OXA-48-like, 16 NDM, 13 VIM genes, and 21 carbapenemase negative isolates. RESULTS: For each carbapenemase, two candidate peptides were validated. Method validation was performed in a blinded manner for all 83 isolates. All carbapenemases were detected. The majority was detected by both target peptides. All target peptides were 100% specific in the tested isolates and no peptide carry-over was detected. CONCLUSION: The applied targeted bottom-up mass spectrometry technique is able to accurately detect the four most prevalent carbapenemases in a single analysis.201931849899
223020.9996Rapid detection of gram-negative antimicrobial resistance determinants directly from positive blood culture broths using a multiplex PCR system. Currently available rapid blood culture diagnostics detect few gram-negative resistance determinants, limiting their clinical utility. We prospectively evaluated the prototype BIOFIRE FILMARRAY Antimicrobial Resistance (AMR) Panel, a rapid multiplex PCR test that detects 31 AMR genes, on residual positive blood culture broths from patients with gram-negative bacteremia due to five target organisms at a New York City hospital. Predicted antimicrobial resistance based on the AMR Panel was compared to results from broth microdilution testing of bloodstream isolates recovered in culture. A simulated stewardship study assessed opportunities for the optimization of therapy if the AMR Panel results had been available for patient care in real time. We enrolled 148 patients with gram-negative bacteremia (Escherichia coli, n = 75; Klebsiella pneumoniae, n = 44; Pseudomonas aeruginosa, n = 17; Enterobacter cloacae complex, n = 9; and Acinetobacter baumannii, n = 3). The sensitivity of the AMR Panel for predicting antimicrobial resistance was ≥90% for 10/14 antimicrobial agents in E. coli and for 10/16 agents in K. pneumoniae. Specificity was ≥90% for 15/17 agents in E. coli and for all 16 agents in K. pneumoniae. Performance for other organisms was poor. For E. coli or K. pneumoniae bacteremia, use of the AMR Panel could have led to earlier escalation or de-escalation of β-lactam therapy in a majority of patients compared to what actually occurred. This study demonstrates that a rapid multiplex PCR test with a large menu of AMR genes can be applied to positive blood culture broths to rapidly predict resistance to frontline antimicrobial agents in patients with E. coli or K. pneumoniae bacteremia.IMPORTANCEPatients with gram-negative bacteremia require urgent treatment with antimicrobial agents that are effective against their infecting pathogen. However, conventional laboratory work-up of blood cultures takes days to yield results, and during this time, patients may receive ineffective therapies. We evaluated the prototype BIOFIRE FILMARRAY AMR Panel, an assay that detects 31 genes in gram-negative bacteria that confer resistance to β-lactams, fluoroquinolones, and aminoglycosides in approximately 1 hour, directly from positive blood culture broths, and compared these results to antimicrobial susceptibility testing of isolates recovered in culture. We found that the AMR Panel accurately predicted resistance in Escherichia coli and Klebsiella pneumoniae to most antimicrobials. Moreover, if results from this assay had been used for patient care, there would have been opportunities to optimize antimicrobial prescribing more quickly than using conventional methods. These data demonstrate how novel molecular assays could optimize care for patients with E. coli and K. pneumoniae bacteremia.202541117625
577930.9996Development of a One-Step Multiplex qPCR Assay for Detection of Methicillin and Vancomycin Drug Resistance Genes in Antibiotic-Resistant Bacteria. The most common antibiotic-resistant bacteria in Korea are methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Pathogen identification in clinical laboratories can be divided into traditional phenotype- and genotype-based methods, both of which are complementary to each other. The genotype-based method using multiplex real-time polymerase chain reaction (PCR) is a rapid and accurate technique that analyzes material at the genetic level by targeting genes simultaneously. Accordingly, we aimed to develop a rapid method for studying the genetic characteristics of antibiotic-resistant bacteria and to provide an experimental guide for the efficient antibiotic resistance gene analysis of mecA detection for MRSA and vanA or vanB detection for VRE using a one-step multiplex qPCR assay at an early stage of infection. As a result, the sensitivity and specificity of the mecA gene for clinical S. aureus isolates, including MRSA and methicillin-susceptible S. aureus, were 97.44% (95% CI, 86.82-99.87%) and 96.15% (95% CI, 87.02-99.32%), respectively. The receiver operating characteristic area under the curve for the diagnosis of MRSA was 0.9798 (*** p < 0.0001). Therefore, the molecular diagnostic method using this newly developed one-step multiplex qPCR assay can provide accurate and rapid results for the treatment of patients with MRSA and VRE infections.202439452724
582740.9996Duplex dPCR System for Rapid Identification of Gram-Negative Pathogens in the Blood of Patients with Bloodstream Infection: A Culture-Independent Approach. Early and accurate detection of pathogens is important to improve clinical outcomes of bloodstream infections (BSI), especially in the case of drug-resistant pathogens. In this study, we aimed to develop a culture-independent digital PCR (dPCR) system for multiplex detection of major sepsiscausing gram-negative pathogens and antimicrobial resistance genes using plasma DNA from BSI patients. Our duplex dPCR system successfully detected nine targets (five bacteria-specific targets and four antimicrobial resistance genes) through five reactions within 3 hours. The minimum detection limit was 50 ag of bacterial DNA, suggesting that 1 CFU/ml of bacteria in the blood can be detected. To validate the clinical applicability, cell-free DNA samples from febrile patients were tested with our system and confirmed high consistency with conventional blood culture. This system can support early identification of some drug-resistant gram-negative pathogens, which can help improving treatment outcomes of BSI.202134528911
583350.9995Rapid identification, virulence analysis and resistance profiling of Staphylococcus aureus by gene segment-based DNA microarrays: application to blood culture post-processing. Up to now, blood culturing systems are the method of choice to diagnose bacteremia. However, definitive pathogen identification from positive blood cultures is a time-consuming procedure, requiring subculture and biochemical analysis. We developed a microarray for the identification of Staphylococcus aureus comprising PCR generated gene-segments, which can reduce the blood culture post-processing time to a single day. Moreover, it allows concomitant identification of virulence factors and antibiotic resistance determinants directly from positive blood cultures without previous amplification by PCR. The assay unambiguously identifies most of the important virulence genes such as tsst-1, sea, seb, eta and antibiotic resistance genes such as mecA, aacA-aphD, blaZ and ermA. To obtain positive signals, 20 ng of purified genomic S. aureus DNA or 2 microg of total DNA extracted from blood culture was required. The microarray specifically distinguished S. aureus from gram-negative bacteria as well as from closely related coagulase negative staphylococci (CoNS). The microarray-based identification of S. aureus can be accomplished on the same day blood cultures become positive in the Bactec. The results of our study demonstrate the feasibility of microarray-based systems for the direct identification and characterization of bacteria from cultured clinical specimens.200717141897
222660.9995Evaluation of the Microbiological Performance and Potential Clinical Impact of New Rapid Molecular Assays for the Diagnosis of Bloodstream Infections. Bloodstream infection (BSI) is a critical medical emergency associated with a high mortality rate. Rapid and accurate identification of the causative pathogen and the results of antimicrobial susceptibility testing are crucial for initiating appropriate antimicrobial therapy. The aim of this study was to evaluate the performance of a new rapid PCR Molecular Mouse System (MMS) for the identification of Gram-negative bacteria (GNB) and GNB resistance genes directly from a positive blood culture (BC). The validation of these rapid multiplex assays was carried out in a real hospital setting. A total of 80 BSI episodes were included in our study and the results were compared with culture-based methods. BC samples in which GNB had previously been detected microscopically and which originated from different hospital wards were analysed. The MMS GNB identification assay achieved a sensitivity of 98.7% and a specificity of 100% for the covered pathogens. In one BC sample, Klebsiella aerogenes was identified at the family level (Enterobacteriaceae) with MMS. However, in three polymicrobial samples, MMS identified bacteria that were not detected by culture-based methods (Klebsiella pneumoniae, K. aerogenes and Stenotrophomonas maltophilia). MMS also showed excellent overall performance in the detection of GNB resistance markers (100% sensitivity and 100% specificity). The type of extended-spectrum beta-lactamase (ESBL) resistance gene identified correctly with MMS was CTX-M-1/9 (n = 17/20), alone or in combination with SHV-type β-lactamase or with the different types of carbapenemase genes. MMS detected one carbapenemase gene of each type (KPC, NDM and OXA-23) and six OXA-48 genes. In addition, the colistin resistance gene mcr-1 was detected in one positive BC with Escherichia coli (E. coli). The time to result was significantly shorter for MMS than for routine culture methods. A retrospective analysis of the patients' medical records revealed that a change in empirical antimicrobial therapy would have been made in around half of the patients following the MMS results. These results support the use of MMS as a valuable complement to conventional culture methods for more rapid BSI diagnosis and adjustment of empirical therapy.202540142509
582970.9995Diagnosing Antibiotic Resistance Using Nucleic Acid Enzymes and Gold Nanoparticles. The rapid and accurate detection of antimicrobial resistance is critical to limiting the spread of infections and delivering effective treatments. Here, we developed a rapid, sensitive, and simple colorimetric nanodiagnostic platform to identify disease-causing pathogens and their associated antibiotic resistance genes within 2 h. The platform can detect bacteria from different biological samples (i.e., blood, wound swabs) with or without culturing. We validated the multicomponent nucleic acid enzyme-gold nanoparticle (MNAzyme-GNP) platform by screening patients with central line associated bloodstream infections and achieved a clinical sensitivity and specificity of 86% and 100%, respectively. We detected antibiotic resistance in methicillin-resistant Staphylococcus aureus (MRSA) in patient swabs with 90% clinical sensitivity and 95% clinical specificity. Finally, we identified mecA resistance genes in uncultured nasal, groin, axilla, and wound swabs from patients with 90% clinical sensitivity and 95% clinical specificity. The simplicity and versatility for detecting bacteria and antibiotic resistance markers make our platform attractive for the broad screening of microbial pathogens.202133970612
224780.9995Metagenomic identification of pathogens and antimicrobial-resistant genes in bacterial positive blood cultures by nanopore sequencing. Nanopore sequencing workflows have attracted increasing attention owing to their fast, real-time, and convenient portability. Positive blood culture samples were collected from patients with bacterial bloodstream infection and tested by nanopore sequencing. This study compared the sequencing results for pathogen taxonomic profiling and antimicrobial resistance genes to those of species identification and phenotypic drug susceptibility using traditional microbiology testing. A total of 37 bacterial positive blood culture results of strain genotyping by nanopore sequencing were consistent with those of mass spectrometry. Among them, one mixed infection of bacteria and fungi was identified using nanopore sequencing and confirmatory quantitative polymerase chain reaction. The amount of sequencing data was 21.89 ± 8.46 MB for species identification, and 1.0 MB microbial strain data enabled accurate determination. Data volumes greater than or equal to 94.6 MB nearly covered all the antimicrobial resistance genes of the bacteria in our study. In addition, the results of the antimicrobial resistance genes were compared with those of phenotypic drug susceptibility testing for Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. Therefore, the nanopore sequencing platform for rapid identification of causing pathogens and relevant antimicrobial resistance genes complementary to conventional blood culture outcomes may optimize antimicrobial stewardship management for patients with bacterial bloodstream infection.202338192400
222590.9995Evaluation of the DNA microarray "AMR Direct Flow Chip Kit" for detection of antimicrobial resistance genes from Gram-positive and Gram-negative bacterial isolated colonies. INTRODUCTION: The AMR Direct Flow Chip assay allows the simultaneous detection of a large variety of antibiotic resistance genetic markers. To assess this kit's performance, we use isolated colonies as starting material. The assay has been approved by the European Economic Area as a suitable device for in vitro diagnosis (CE IVD) using clinical specimens. METHODS: A total of 210 bacterial isolates harbouring either one or more antimicrobial resistance genes including plasmid-encoded extended-spectrum β-lactamases (SHV, CTX-M) and carbapenemases (GES, SME, KPC, NMC/IMI, SIM, GIM, SPM, NDM, VIM, IMP, and OXA), mecA, vanA and vanB, and 30 controls were included. RESULTS: The assay displayed a sensitivity and specificity of 100% for all target genes included in the array. CONCLUSION: The AMR Direct Flow Chip Kit is an accurate assay for detecting genes which commonly confer resistance to β-lactams and vancomycin from isolated colonies in culture of Gram-positive and Gram-negative bacteria.201930857832
1482100.9995Evaluation and clinical practice of pathogens and antimicrobial resistance genes of BioFire FilmArray Pneumonia panel in lower respiratory tract infections. BACKGROUND: Existing panels for lower respiratory tract infections (LRTIs) are slow and lack quantification of important pathogens and antimicrobial resistance, which are not solely responsible for their complex etiology and antibiotic resistance. BioFire FilmArray Pneumonia (PN) panels may provide rapid information on their etiology. METHODS: The bronchoalveolar lavage fluid of 187 patients with LRTIs was simultaneously analyzed using a PN panel and cultivation, and the impact of the PN panel on clinical practice was assessed. The primary endpoint was to compare the consistency between the PN panel and conventional microbiology in terms of etiology and drug resistance, as well as to explore the clinical significance of the PN panel. The secondary endpoint was pathogen detection using the PN panel in patients with community-acquired pneumonia (CAP) or hospital-acquired pneumonia (HAP). RESULTS: Fifty-seven patients with HAP and 130 with CAP were included. The most common pathogens of HAP were Acinetobacter baumannii and Klebsiella pneumoniae, with the most prevalent antimicrobial resistance (AMR) genes being CTX-M and KPC. For CAP, the most common pathogens were Haemophilus influenzae and Staphylococcus aureus, with the most frequent AMR genes being CTX-M and VIM. Compared with routine bacterial culture, the PN panel demonstrated an 85% combined positive percent agreement (PPA) and 92% negative percent agreement (NPA) for the qualitative identification of 13 bacterial targets. PN detection of bacteria with higher levels of semi-quantitative bacteria was associated with more positive bacterial cultures. Positive concordance between phenotypic resistance and the presence of corresponding AMR determinants was 85%, with 90% positive agreement between CTX-M-type extended-spectrum beta-lactamase gene type and phenotype and 100% agreement for mecA/C and MREJ. The clinical benefit of the PN panel increased by 25.97% compared with traditional cultural tests. CONCLUSION: The bacterial pathogens and AMR identified by the PN panel were in good agreement with conventional cultivation, and the clinical benefit of the PN panel increased by 25.97% compared with traditional detection. Therefore, the PN panel is recommended for patients with CAP or HAP who require prompt pathogen diagnosis and resistance identification.202438123753
5041110.9995Development and Validation of a Clinical Laboratory Improvement Amendments-Compliant Multiplex Real-Time PCR Assay for Detection of mcr Genes. Increased use of colistin in both human and veterinary medicine has led to the emergence of plasmid-mediated colistin resistance (mcr genes). In this study, we report the development of a real-time PCR assay using TaqMan probe-based chemistry for detection of mcr genes from bacterial isolates. Positive control isolates harboring mcr-1 and mcr-2 yielded exponential amplification curves with the assay, and the amplification efficiency was 98% and 96% for mcr-1 and mcr-2, respectively. Each target gene could be reproducibly detected from a sample containing 10(3) cfu/mL of mcr-harboring bacteria, and there was no cross-reactivity with DNA extracted from several multidrug-resistant bacteria harboring other resistance genes, but lacking mcr genes. Both sensitivity and specificity of the mcr real-time PCR assay were 100% in a method validation performed with a set of 25 previously well-characterized bacterial isolates containing mcr-positive and -negative bacteria. This newly developed assay is a rapid and sensitive tool for detecting emerging mcr genes in cultured bacterial isolates. The assay was successfully validated according to quality standards of the Clinical Laboratory Improvement Amendments (CLIA).201930942652
5796120.9995Antibiotic treatment algorithm development based on a microarray nucleic acid assay for rapid bacterial identification and resistance determination from positive blood cultures. Rapid diagnosis of bloodstream infections remains a challenge for the early targeting of an antibiotic therapy in sepsis patients. In recent studies, the reliability of the Nanosphere Verigene Gram-positive and Gram-negative blood culture (BC-GP and BC-GN) assays for the rapid identification of bacteria and resistance genes directly from positive BCs has been demonstrated. In this work, we have developed a model to define treatment recommendations by combining Verigene test results with knowledge on local antibiotic resistance patterns of bacterial pathogens. The data of 275 positive BCs were analyzed. Two hundred sixty-three isolates (95.6%) were included in the Verigene assay panels, and 257 isolates (93.5%) were correctly identified. The agreement of the detection of resistance genes with subsequent phenotypic susceptibility testing was 100%. The hospital antibiogram was used to develop a treatment algorithm on the basis of Verigene results that may contribute to a faster patient management.201626712265
5785130.9995Molecular characterization of resistance and biofilm genes of ESKAPE pathogens isolated from clinical samples: examination of the effect of boric acid on biofilm ability by cell culture method. Biofilm formation ranks first among the resistance and virulence factors crucial in forming ESKAPE pathogens. Once biofilm is formed, treating the infection with existing drugs is often futile. Therefore, in this study, resistant ESKAPE pathogens were isolated from intensive care units and sent to Atatürk University Yakutiye Research Hospital Microbiology Laboratory. This study investigated the biofilm formation and molecular characterization of resistant ESKAPE pathogens isolated from intensive care units. The bacteria's biofilm formation abilities, genes responsible for biofilm formation, and resistance characteristics were identified. The effect of boric acid (BA) on resistance and bacterial genes was evaluated by a bacterial infection cell culture model. The highest biofilm formation was observed in Escherichia coli, Enterococcus spp., and Pseudomonas aeruginosa Enterococcus spp. isolates showed the vanA gene in 14.6% and the vanC gene in 61% of the samples. Among Staphylococcus spp. isolates, 48.3% were MSSA, 34.5% were MRCNS, and 17.2% were MRSA. The KPC gene was detected in 50%, the OXA-48 gene in 40%, and the NDM gene in 15% of the isolates. In P. aeruginosa, the LasI and LasR quorum sensing system genes were found in 38.5% and 30.8% of the isolates, respectively. In E. coli isolates, OXA-48 was present in 35%, KPC in 31.7%, and TEM in 12.5%. BA demonstrated significant activity against ESKAPE pathogens. The combined antimicrobial activity of boron compounds showed a decrease in the expression level of the resistance gene. It will be promising for preventing hospital-associated infections.202540025436
2229140.9995A pentaplex real-time PCR assay for rapid identification of major beta-lactamase genes KPC, NDM, CTX, CMY, and OXA-48 directly from bacteria in blood. Introduction. Antibiotic resistance, particularly in cases of sepsis, has emerged as a growing global public health concern and economic burden. Current methods of blood culture and antimicrobial susceptibility testing of agents involved in sepsis can take as long as 3-5 days. It is vital to rapidly identify which antimicrobials can be used to effectively treat sepsis cases on an individual basis. Here, we present a pentaplex, real-time PCR-based assay that can quickly identify the most common beta-lactamase genes (Klebsiella pneumoniae carbapenemase (KPC); New Delhi metallo-beta-lactamase (NDM); cefotaximase-Munich (CTX-M); cephamycin AmpC beta-lactamases (CMY); and Oxacillinase-48 (OXA-48)) from pathogens derived directly from the blood of patients presenting with bacterial septicemia.Aim. To develop an assay which can rapidly identify the most common beta-lactamase genes in Carbapenem-resistant Enterobacteriaceae bacteria (CREs) from the United States.Hypothesis/Gap Statement. Septicemia caused by carbapenem-resistant bacteria has a death rate of 40-60 %. Rapid diagnosis of antibiotic susceptibility directly from bacteria in blood by identification of beta-lactamase genes will greatly improve survival rates. In this work, we develop an assay capable of concurrently identifying the five most common beta-lactamase and carbapenemase genes.Methodology. Primers and probes were created which can identify all subtypes of Klebsiella pneumoniae carbapenemase (KPC); New Delhi metallo-beta-lactamase (NDM); cefotaximase-Munich (CTX); cephamycin AmpC beta-lactamase (CMY); and oxacillinase-48 (OXA-48). The assay was validated using 13 isolates containing various PCR targets from the Centre for Disease Control Antimicrobial Resistance Isolate Bank Enterobacterales Carbapenemase Diversity Panel. Blood obtained from volunteers was spiked with CREs and bacteria were separated, lysed, and subjected to analysis via the pentaplex assay.Results. This pentaplex assay successfully identified beta-lactamase genes derived from bacteria separated from blood at concentrations of 4-8 c.f.u. ml(-1).Conclusion. This assay will improve patient outcomes by supplying physicians with critical drug resistance information within 2 h of septicemia onset, allowing them to prescribe effective antimicrobials corresponding to the resistance gene(s) present in the pathogen. In addition, information supplied by this assay will lessen the inappropriate use of broad-spectrum antimicrobials and prevent the evolution of further antibiotic resistance.202134878374
5694150.9995Multiplex characterization of human pathogens including species and antibiotic-resistance gene identification. The efficient medical treatment of infections requires detailed information about the pathogens involved and potential antibiotic-resistance mechanisms. The dramatically increasing incidence of multidrug-resistant bacteria especially highlights the importance of sophisticated diagnostic tests enabling a fast patient-customized therapy. However, the current molecular detection methods are limited to either the detection of species or only a few antibiotic-resistance genes.In this work, we present a human pathogen characterization assay using a rRNA gene microarray identifying 75 species comprising bacteria and fungi. A statistical classifier was developed to facilitate the automated species identification. Additionally, the clinically most important β-lactamases were identified simultaneously in a 100-plex reaction using padlock probes and the same microarray. The specificity and sensitivity of the combined assay was determined using clinical isolates. The detection limit was 10(5) c.f.u. ml(-1), recovering 89 % of the detectable β-lactamase-encoding genes specifically. The total assay time was less than 7 hand the modular character of the antibiotic-resistance detection allows the easy integration of further genetic targets. In summary, we present a fast, highly specific and sensitive multiplex pathogen characterization assay.201626489938
2235160.9995Nanosphere's Verigene(®) Blood Culture Assay to Detect Multidrug-Resistant Gram-Negative Bacterial Outbreak: A Prospective Study on 79 Hematological Patients in a Country with High Prevalence of Antimicrobial Resistance. Infections are a major cause of morbidity and mortality in hematological patients. We prospectively tested a new molecular assay (Verigene(®)) in 79 consecutive hematological patients, with sepsis by gram-negative bacteria. A total of 82 gram-negative microorganisms were isolated by blood cultures, of which 76 cases were mono-microbial. Considering the bacteria detectable by the system, the concordance with standard blood cultures was 100%. Resistance genes were detected in 20 of the isolates and 100% were concordant with the phenotypic antibiotic resistance. Overall, this new assay correctly identified 66/82 of all the gram-negative pathogens, yielding a general sensitivity of 80.5%, and providing information on genetic antibiotic resistance in a few hours. This new molecular assay could ameliorate patient management, resulting in a more rational use of antibiotics.201934595420
2236170.9995Development of a Multiplex PCR Platform for the Rapid Detection of Bacteria, Antibiotic Resistance, and Candida in Human Blood Samples. The diagnosis of bloodstream infections (BSIs) still relies on blood culture (BC), but low turnaround times may hinder the early initiation of an appropriate antimicrobial therapy, thus increasing the risk of infection-related death. We describe a direct and rapid multiplex PCR-based assay capable of detecting and identifying 16 bacterial and four Candida species, as well as three antibiotic-resistance determinants, in uncultured samples. Using whole-blood samples spiked with microorganisms at low densities, we found that the MicrobScan assay had a mean limit of detection of 15.1 ± 3.3 CFU of bacteria/Candida per ml of blood. When applied to positive BC samples, the assay allowed the sensitive and specific detection of BSI pathogens, including bla(KPC)-, mecA-, or vanA/vanB-positive bacteria. We evaluated the assay using prospectively collected blood samples from patients with suspected BSI. The sensitivity and specificity were 86.4 and 97.0%, respectively, among patients with positive BCs for the microorganisms targeted by the assay or patients fulfilling the criteria for infection. The mean times to positive or negative assay results were 5.3 ± 0.2 and 5.1 ± 0.1 h, respectively. Fifteen of 20 patients with MicrobScan assay-positive/BC-negative samples were receiving antimicrobial therapy. In conclusion, the MicrobScan assay is well suited to complement current diagnostic methods for BSIs.201931799215
5797180.9995PCR-reverse blot hybridization assay for screening and identification of pathogens in sepsis. Rapid and accurate identification of the pathogens involved in bloodstream infections is crucial for the prompt initiation of appropriate therapy, as this can decrease morbidity and mortality rates. A PCR-reverse blot hybridization assay for sepsis, the reverse blot hybridization assay (REBA) Sepsis-ID test, was developed; it uses pan-probes to distinguish Gram-positive and -negative bacteria and fungi. In addition, the assay was designed to identify bacteria and fungi using six genus-specific and 13 species-specific probes; it uses additional probes for antibiotic resistance genes, i.e., the mecA gene of methicillin-resistant Staphylococcus aureus (MRSA) and the vanA and vanB genes of vancomycin-resistant enterococci (VRE). The REBA Sepsis-ID test successfully identified clinical isolates and blood culture samples as containing Gram-positive bacteria, Gram-negative bacteria, or fungi. The results matched those obtained with conventional microbiological methods. For the REBA Sepsis-ID test, of the 115 blood culture samples tested, 47 (40.8%) and 49 (42.6%) samples were identified to the species and genus levels, respectively, and the remaining 19 samples (16.5%), which included five Gram-positive rods, were identified as Gram-positive bacteria, Gram-negative bacteria, or fungi. The antibiotic resistances of the MRSA and VRE strains were identified using both conventional microbiological methods and the REBA Sepsis-ID test. In conclusion, the REBA Sepsis-ID test developed for this study is a fast and reliable test for the identification of Gram-positive bacteria, Gram-negative bacteria, fungi, and antibiotic resistance genes (including mecA for MRSA and the vanA and vanB genes for VRE) in bloodstream infections.201323447637
2240190.9995Evaluation of multiplex tandem PCR (MT-PCR) assays for the detection of bacterial resistance genes among Enterobacteriaceae in clinical urines. BACKGROUND: Increasing resistance drives empirical use of less potent and previously reserved antibiotics, including for urinary tract infections (UTIs). Molecular profiling, without culture, might better guide early therapy. OBJECTIVES: To explore the potential of AusDiagnostics multiplex tandem (MT) PCR UTI assays. METHODS: Two MT-PCR assays were developed successively, seeking 8 or 16 resistance genes. Amplification was tracked in real time, with melting temperatures used to confirm product identity. Assays were variously performed on: (i) extracted DNA; (ii) cultured bacteria; (iii) urine spiked with reference strains; and (iv) bacteria harvested from clinical urines. Results were compared with those from sequencing, real-time SybrGreen PCR or phenotypic susceptibility. RESULTS: Performance was similar irrespective of whether DNA, cultures or urines were used, with >90% sensitivity and specificity with respect to common β-lactamases, dfr genes and aminoglycoside resistance determinants except aadA1/A2/A3, for which carriage correlated poorly with streptomycin resistance. Fluoroquinolone-susceptible and -resistant Escherichia coli (but not other species) were distinguished by the melting temperatures of their gyrA PCR products. The time from urine to results was <3 h. CONCLUSIONS: The MT-PCR assays rapidly identified resistance genes from Gram-negative bacteria in urines as well as from cultivated bacteria. Used directly on urines, this assay has the potential to guide early therapy.201930476137