scholarly journals Duplex real-time PCR assay for the simultaneous detection of Achromobacter xylosoxidans and Achromobacter spp.

2020 ◽  
Vol 6 (7) ◽  
Author(s):  
Erin P. Price ◽  
Valentina Soler Arango ◽  
Timothy J. Kidd ◽  
Tamieka A. Fraser ◽  
Thuy-Khanh Nguyen ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Type Species ◽  
Simultaneous Detection ◽  
Diagnostic Methods ◽  
Achromobacter Xylosoxidans ◽  
Resistant Bacteria ◽  
Pcr Assay ◽  
Content Type ◽  
Link Type

Several members of the Gram-negative environmental bacterial genus Achromobacter are associated with serious infections, with Achromobacter xylosoxidans being the most common. Despite their pathogenic potential, little is understood about these intrinsically drug-resistant bacteria and their role in disease, leading to suboptimal diagnosis and management. Here, we performed comparative genomics for 158 Achromobacter spp. genomes to robustly identify species boundaries, reassign several incorrectly speciated taxa and identify genetic sequences specific for the genus Achromobacter and for A. xylosoxidans . Next, we developed a Black Hole Quencher probe-based duplex real-time PCR assay, Ac-Ax, for the rapid and simultaneous detection of Achromobacter spp. and A. xylosoxidans from both purified colonies and polymicrobial clinical specimens. Ac-Ax was tested on 119 isolates identified as Achromobacter spp. using phenotypic or genotypic methods. In comparison to these routine diagnostic methods, the duplex assay showed superior identification of Achromobacter spp. and A. xylosoxidans , with five Achromobacter isolates failing to amplify with Ac-Ax confirmed to be different genera according to 16S rRNA gene sequencing. Ac-Ax quantified both Achromobacter spp. and A. xylosoxidans down to ~110 genome equivalents and detected down to ~12 and ~1 genome equivalent(s), respectively. Extensive in silico analysis, and laboratory testing of 34 non- Achromobacter isolates and 38 adult cystic fibrosis sputa, confirmed duplex assay specificity and sensitivity. We demonstrate that the Ac-Ax duplex assay provides a robust, sensitive and cost-effective method for the simultaneous detection of all Achromobacter spp. and A. xylosoxidans and will facilitate the rapid and accurate diagnosis of this important group of pathogens.

scholarly journals Duplex real-time PCR assay for the simultaneous detection of Achromobacter xylosoxidans and Achromobacter spp

2020 ◽  
Author(s):  
Erin P. Price ◽  
Valentina Soler Arango ◽  
Timothy J. Kidd ◽  
Tamieka A. Fraser ◽  
Thuy-Khanh Nguyen ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Diagnostic Methods ◽  
Achromobacter Xylosoxidans ◽  
Resistant Bacteria ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Pathogenic Potential ◽  
Specificity And Sensitivity

AbstractSeveral members of the Gram-negative environmental bacterial genus, Achromobacter, are associated with serious infections in immunocompromised individuals, of which Achromobacter xylosoxidans is the most common. Despite their pathogenic potential, comparatively little is understood about these intrinsically drug-resistant bacteria and their role in disease, leading to suboptimal diagnosis and management of Achromobacter infections. Here, we performed comparative genomics of 158 Achromobacter spp. genomes to robustly identify species boundaries, to reassign several incorrectly speciated taxa, and to identify genetic sequences specific for the Achromobacter genus and for A. xylosoxidans. Next, we developed a Black Hole Quencher probe-based duplex real-time PCR assay, Ac-Ax, for the rapid and simultaneous detection of Achromobacter spp. and A. xylosoxidans from both purified colonies and polymicrobial clinical specimens. Ac-Ax was tested on 119 isolates identified as Achromobacter spp. using phenotypic or genotypic methods. In comparison to these routine diagnostic methods, the duplex assay showed superior identification of Achromobacter spp. and A. xylosoxidans, with five Achromobacter isolates failing to amplify with Ac-Ax confirmed to be different genera according to 16S rRNA gene sequencing. Ac-Ax quantified both Achromobacter spp. and A. xylosoxidans down to ∼110 genome equivalents, and detected down to ∼12 and ∼1 genome equivalent/s, respectively. In silico analysis, and laboratory testing of 34 non-Achromobacter isolates and 38 adult CF sputa, confirmed duplex assay specificity and sensitivity. We demonstrate that the Ac-Ax duplex assay provides a robust, sensitive, and cost-effective method for the simultaneous detection of all Achromobacter spp. and A. xylosoxidans, and will facilitate the rapid and accurate diagnosis of this important group of pathogens.

scholarly journals A Real-Time PCR Assay for Simultaneous Detection and Differentiation of Four Common Entamoeba Species That Infect Humans

2020 ◽  
Vol 59 (1) ◽  
pp. e01986-20
Author(s):  
Ibne Karim M. Ali ◽  
Shantanu Roy
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Small Subunit ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Content Type ◽  
Rdna Sequences ◽  
Appropriate Treatment

ABSTRACTThere are over 40 species within the genus Entamoeba, eight of which infect humans. Of these, four species (Entamoeba histolytica, E. dispar, E. moshkovskii, and E. bangladeshi) are morphologically indistinguishable from each other, and yet differentiation is important for appropriate treatment decisions. Here, we developed a hydrolysis probe-based tetraplex real-time PCR assay that can simultaneously detect and differentiate these four species in clinical samples. In this assay, multicopy small-subunit (SSU) ribosomal DNA (rDNA) sequences were used as targets. We determined that the tetraplex real-time PCR can detect amebic DNA corresponding to as little as a 0.1 trophozoite equivalent of any of these species. We also determined that this assay can detect E. histolytica DNA in the presence of 10-fold more DNA from another Entamoeba species in mixed-infection scenarios. With a panel of more than 100 well-characterized clinical samples diagnosed and confirmed using a previously published duplex real-time PCR (capable of detecting E. histolytica and E. dispar), our tetraplex real-time PCR assay demonstrated levels of sensitivity and specificity comparable with those demonstrated by the duplex real-time PCR assay. The advantage of our assay over the duplex assay is that it can specifically detect two additional Entamoeba species and can be used in conventional PCR format. This newly developed assay will allow further characterization of the epidemiology and pathogenicity of the four morphologically identical Entamoeba species, especially in low-resource settings.

A 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

2021 ◽  
Vol 70 (12) ◽  
Author(s):  
Taalin R. Hoj ◽  
Bradley McNeely ◽  
Kylie Webber ◽  
Evelyn Welling ◽  
William G. Pitt ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Klebsiella Pneumoniae ◽  
Real Time ◽  
Real Time Pcr ◽  
Type Species ◽  
New Delhi ◽  
Beta Lactamase ◽  
Content Type ◽  
Link Type ◽  
Carbapenem Resistant

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.

scholarly journals A Real-Time Multiplex PCR Assay for Detection ofElizabethkingiaSpecies and Differentiation betweenElizabethkingia anophelisandE. meningoseptica

2019 ◽  
Vol 57 (4) ◽  
Author(s):  
Aubree J. Kelly ◽  
Sandor E. Karpathy ◽  
Christopher A. Gulvik ◽  
Melissa L. Ivey ◽  
Anne M. Whitney ◽  
...  
Keyword(s):  
Comparative Genomics ◽  
Real Time ◽  
Nosocomial Infections ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Diagnostic Tools ◽  
Pcr Assay ◽  
Content Type ◽  
Multiplex Pcr Assay ◽  
Elizabethkingia Meningoseptica

ABSTRACTNosocomial infections ofElizabethkingiaspecies can have fatal outcomes if not identified and treated properly. The current diagnostic tools available require culture and isolation, which can extend the reporting time and delay treatment. Using comparative genomics, we developed an efficient multiplex real-time PCR for the simultaneous detection of all known species ofElizabethkingia, as well as differentiating the two most commonly reported species,Elizabethkingia anophelisandElizabethkingia meningoseptica.

scholarly journals Rapid nanopore-based DNA sequencing protocol of antibiotic-resistant bacteria for use in surveillance and outbreak investigation

2021 ◽  
Vol 7 (4) ◽  
Author(s):  
Fabienne Antunes Ferreira ◽  
Karin Helmersen ◽  
Tina Visnovska ◽  
Silje Bakken Jørgensen ◽  
Hege Vangstein Aamot
Keyword(s):  
Phylogenetic Analysis ◽  
Real Time ◽  
Type Species ◽  
Outbreak Investigation ◽  
Complete Analysis ◽  
Resistant Bacteria ◽  
Antibiotic Resistant ◽  
Content Type ◽  
Link Type ◽  
Outbreak Investigations

Outbreak investigations are essential to control and prevent the dissemination of pathogens. This study developed and validated a complete analysis protocol for faster and more accurate surveillance and outbreak investigations of antibiotic-resistant microbes based on Oxford Nanopore Technologies (ONT) DNA whole-genome sequencing. The protocol was developed using 42 methicillin-resistant Staphylococcus aureus (MRSA) isolates identified from former well-characterized outbreaks. The validation of the protocol was performed using Illumina technology (MiSeq, Illumina). Additionally, a real-time outbreak investigation of six clinical S. aureus isolates was conducted to test the ONT-based protocol. The suggested protocol includes: (1) a 20 h sequencing run; (2) identification of the sequence type (ST); (3) de novo genome assembly; (4) polishing of the draft genomes; and (5) phylogenetic analysis based on SNPs. After the sequencing run, it was possible to identify the ST in 2 h (20 min per isolate). Assemblies were achieved after 4 h (40 min per isolate) while the polishing was carried out in 7 min per isolate (42 min in total). The phylogenetic analysis took 0.6 h to confirm an outbreak. Overall, the developed protocol was able to at least discard an outbreak in 27 h (mean) after the bacterial identification and less than 33 h to confirm it. All these estimated times were calculated considering the average time for six MRSA isolates per sequencing run. During the real-time S. aureus outbreak investigation, the protocol was able to identify two outbreaks in less than 31 h. The suggested protocol enables identification of outbreaks in early stages using a portable and low-cost device along with a streamlined downstream analysis, therefore having the potential to be incorporated in routine surveillance analysis workflows. In addition, further analysis may include identification of virulence and antibiotic resistance genes for improved pathogen characterization.

scholarly journals Evaluation of the New BD Max GC Real-Time PCR Assay, Analytically and Clinically as a Supplementary Test for the BD ProbeTec GC Qx Amplified DNA Assay, for Molecular Detection of Neisseria gonorrhoeae

2015 ◽  
Vol 53 (12) ◽  
pp. 3935-3937 ◽  
Author(s):  
Daniel Golparian ◽  
Stina Boräng ◽  
Martin Sundqvist ◽  
Magnus Unemo
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Real Time ◽  
Sensitivity And Specificity ◽  
Real Time Pcr ◽  
Molecular Detection ◽  
Analytical Sensitivity ◽  
Pcr Assay ◽  
Content Type ◽  
Dna Assay ◽  
Supplementary Test

The new BD Max GC real-time PCR assay showed high clinical and analytical sensitivity and specificity. It can be an effective and accurate supplementary test for the BD ProbeTec GC Qx amplified DNA assay, which had suboptimal specificity, and might also be used for initial detection ofNeisseria gonorrhoeae.

Adaptation of gltA and ssrA assays for diversity profiling by Illumina sequencing to identify Bartonella henselae, B. clarridgeiae and B. koehlerae

2021 ◽  
Vol 70 (7) ◽  
Author(s):  
Rosemonde Isabella Power ◽  
Nichola Elisa Davies Calvani ◽  
Yaarit Nachum-Biala ◽  
Harold Salant ◽  
Shimon Harrus ◽  
...  
Keyword(s):  
Illumina Sequencing ◽  
Type Species ◽  
Bacterial Infections ◽  
Sanger Sequencing ◽  
Bartonella Henselae ◽  
Diagnostic Methods ◽  
Mixed Infections ◽  
Accurate Identification ◽  
Content Type ◽  
Link Type

Introduction. Bartonellosis is an emerging zoonotic disease caused by bacteria of the genus Bartonella . Mixed Bartonella infections are a well-documented phenomenon in mammals and their ectoparasites. The accurate identification of Bartonella species in single and mixed infections is valuable, as different Bartonella species have varying impacts on infected hosts. Gap Statement. Current diagnostic methods are inadequate at identifying the Bartonella species present in mixed infections. Aim. The aim of this study was to adopt a Next Generation Sequencing (NGS) approach using Illumina sequencing technology to identify Bartonella species and demonstrate that this approach can resolve mixed Bartonella infections. Methodology. We used Illumina PCR amplicon NGS to target the ssrA and gltA genes of Bartonella in fleas collected from cats, dogs and a hedgehog in Israel. We included artificially mixed Bartonella samples to demonstrate the ability for NGS to resolve mixed infections and we compared NGS to traditional Sanger sequencing. Results. In total, we identified 74 Ctenocephalides felis, two Ctenocephalides canis, two Pulex irritans and three Archaeopsylla e. erinacei fleas. Real-time PCR of a subset of 48 fleas revealed that twelve were positive for Bartonella , all of which were cat fleas. Sanger sequencing of the ssrA and gltA genes confirmed the presence of Bartonella henselae , Bartonella clarridgeiae and Bartonella koehlerae . Illumina NGS of ssrA and gltA amplicons further confirmed the Bartonella species identity in all 12 flea samples and unambiguously resolved the artificially mixed Bartonella samples. Conclusion. The adaptation and multiplexing of existing PCR assays for diversity profiling via NGS is a feasible approach that is superior to traditional Sanger sequencing for Bartonella speciation and resolving mixed Bartonella infections. The adaptation of other PCR primers for Illumina NGS will be useful in future studies where mixed bacterial infections may be present.

Improved diagnosis of gastrointestinal infections using a semi-automated multiplex real-time PCR for detection of enteropathogens

2021 ◽  
Vol 70 (9) ◽  
Author(s):  
Berta Fidalgo ◽  
Elisa Rubio ◽  
Victor Pastor ◽  
Marta Parera ◽  
Clara Ballesté-Delpierre ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Enteric Pathogens ◽  
Pcr Analysis ◽  
Culture Methods ◽  
Gastrointestinal Infections ◽  
Content Type ◽  
Link Type ◽  
Microbiological Culture ◽  
Micro Organisms

Introduction. The identification of enteropathogens is critical for the clinical management of patients with suspected gastrointestinal infection. The FLOW multiplex PCR system (FMPS) is a semi-automated platform (FLOW System, Roche) for multiplex real-time PCR analysis. Hypothesis/Gap Statement. FMPS has greater sensitivity for the detection of enteric pathogens than standard methods such as culture, biochemical identification, immunochromatography or microscopic examination. Aim.The diagnostic performance of the FMPS was evaluated and compared to that of traditional microbiological procedures. Methodology. A total of 10 659 samples were collected and analysed over a period of 7 years. From 2013 to 2018 (every July to September), samples were processed using standard microbiological culture methods. In 2019, the FMPS was implemented using real-time PCR to detect the following enteropathogens: Shigella spp., Salmonella spp., Campylobacter spp., Giardia intestinalis, Entamoeba histolytica, Blastocystis hominis, Cryptosporidum spp., Dientamoeba fragilis, adenovirus, norovirus and rotavirus. Standard microbiological culture methods (2013–2018) included stool culture, microscopy and immunochromatography. Results. A total of 1078 stool samples were analysed prospectively using the FMPS from July to September (2019): bacterial, parasitic and viral pathogens were identified in 15.3, 9.71 and 5.29 % of cases, respectively. During the same period of 6 years (2013–2018), the proportion of positive identifications using standard microbiological methods from 2013 to 2018 was significantly lower. A major significant recovery improvement was observed for all bacteria species tested: Shigella spp./enteroinvasive Escherichia coli (EIEC) (P <0.05), Salmonella spp. (P <0.05) and Campylobacter spp. (P <0.05). Marked differences were also observed for the parasites G. intestinalis, Cryptosporidium spp. and D. fragilis. Conclusion. These results support the value of multiplex real-time PCR analysis for the detection of enteric pathogens in laboratory diagnosis with outstanding performance in identifying labile micro-organisms. The identification of unsuspected micro-organisms for less specific clinical presentations may also impact on clinical practice and help optimize patient management.

scholarly journals Duplex Real-Time PCR Assay for Rapid Detection of Ampicillin-Resistant Enterococcus faecium

2004 ◽  
Vol 48 (2) ◽  
pp. 556-560 ◽  
Author(s):  
Stein Christian Mohn ◽  
Arve Ulvik ◽  
Roland Jureen ◽  
Rob J. L. Willems ◽  
Janetta Top ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Enterococcus Faecium ◽  
Rapid Detection ◽  
Resistant Bacteria ◽  
Pcr Assay ◽  
Culture Methods ◽  
Accurate Identification ◽  
Antibiotic Resistant ◽  
Highly Correlated

ABSTRACT Rapid and accurate identification of carriers of resistant microorganisms is an important aspect of efficient infection control in hospitals. Traditional identification methods of antibiotic-resistant bacteria usually take at least 3 to 4 days after sampling. A duplex real-time PCR assay was developed for rapid detection of ampicillin-resistant Enterococcus faecium (ARE). Primers and probes that are used in this assay specifically detected the d-Ala-d-Ala ligase gene of E. faecium and the modified penicillin-binding protein 5 gene (pbp5) carrying the Glu-to-Val substitution at position 629 (Val-629) in a set of 129 tested E. faecium strains with known pbp5 sequence. Presence of the Val-629 in the strain set from 11 different countries was highly correlated with ampicillin resistance. In a screening of hospitalized patients, the real-time PCR assay yielded a sensitivity and a specificity for the detection of ARE colonization of 95% and 100%, respectively. The results were obtained 4 h after samples were harvested from overnight broth of rectal swab samples, identifying both species and the resistance marker mutation in pbp5. This novel assay reliably identifies ARE 2 to 3 days more quickly than traditional culture methods, thereby increasing laboratory throughput, making it useful for rectal screening of ARE. The assay demonstrates the advantages of real-time PCR for detection of nosocomial pathogens.

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