Loop-mediated isothermal amplification assays for the detection of antimicrobial resistance elements in Vibrio cholera.

Background: The bacterium Vibrio cholerae causes diarrheal illness and can acquire genetic material leading to multiple drug resistance (MDR). Rapid detection of resistance-conferring mobile genetic elements helps avoid the prescription of ineffective antibiotics for specific strains. Colorimetric loop-mediated isothermal amplification (LAMP) assays provide a rapid and cost-effective means for detection at point-of-care since they do not require specialized equipment, require limited expertise to perform, and can take less than 30 min to perform in resource limited regions.

Performance of rapid antigen tests to detect SARS-CoV-2 variant diversity and correlation with viral culture positivity: implication for diagnostic development and future public health strategies.

Unlabelled: Antigen-based rapid diagnostic tests (Ag-RDTs) provide timely results, are simple to use, and are less expensive than molecular assays. Recent studies suggest that antigen-based testing aligns with virus culture-based results (a proxy of contagiousness at the peak viral phase of illness); however, the performance of Ag-RDTs for newer SARS-CoV-2 variants is unclear. In this study, we (i) assessed the performance of Ag-RDTs and diagnostic antibodies to detect a range of SARS-CoV-2 variants and (ii) determined whether Ag-RDT results correlated with culture positivity.

Wastewater Surveillance Pilot at US Military Installations: Cost Model Analysis.

Background: The COVID-19 pandemic highlighted the need for pathogen surveillance systems to augment both early warning and outbreak monitoring/control efforts. Community wastewater samples provide a rapid and accurate source of environmental surveillance data to complement direct patient sampling. Due to its global presence and critical missions, the US military is a leader in global pandemic preparedness efforts.

Impact of template denaturation prior to whole genome amplification on gene detection in high GC-content species, Burkholderia mallei and B. pseudomallei.

Objective: In this study, we sought to determine the types and prevalence of antimicrobial resistance determinants (ARDs) in Burkholderia spp. strains using the Antimicrobial Resistance Determinant Microarray (ARDM).

Results: Whole genome amplicons from 22 B.

Amplicon Sequencing Minimal Information (ASqMI): Quality and Reporting Guidelines for Actionable Calls in Biodefense Applications.

Background: Accurate, high-confidence data is critical for assessing potential biothreat incidents. In a biothreat event, false-negative and -positive results have serious consequences. Worst case scenarios can result in unnecessary shutdowns or fatalities at an exorbitant monetary and psychological cost, respectively.

Draft Genome Assemblies of Phage AP50c-Resistant Derivatives of Bacillus anthracis Sterne Strain 7702 Lacking Plasmid pXO2.

Mutants of the attenuated Bacillus anthracis (Sterne) strain 7702 that are resistant to phage AP50c have been previously described. Here, we report the draft genome assemblies of the parent strain, several phage-resistant derivatives, and mutants of genes in the pathways for synthesis and assembly of the S-layer.

Development, testing and validation of a SARS-CoV-2 multiplex panel for detection of the five major variants of concern on a portable PCR platform.

Many SARS-CoV-2 variants have emerged during the course of the COVID-19 pandemic. These variants have acquired mutations conferring phenotypes such as increased transmissibility or virulence, or causing diagnostic, therapeutic, or immune escape. Detection of Alpha and the majority of Omicron sublineages by PCR relied on the so-called S gene target failure due to the deletion of six nucleotides coding for amino acids 69-70 in the spike (S) protein.

Optimization of Oxford Nanopore Technology Sequencing Workflow for Detection of Amplicons in Real Time Using ONT-DART Tool.

An optimized, well-tested and validated targeted genomic sequencing-based high-throughput assay is currently not available ready for routine biodefense and biosurveillance applications. Earlier, we addressed this gap by developing and establishing baseline comparisons of a multiplex end-point Polymerase Chain Reaction (PCR) assay followed by Oxford Nanopore Technology (ONT) based amplicon sequencing to real time PCR and customized data processing. Here, we expand upon this effort by identifying the optimal ONT library preparation method for integration into a novel software platform ONT-DART (ONT-Detection of Amplicons in Real-Time).

Impact of SARS-CoV-2 Mutations on PCR Assay Sequence Alignment.

Real-time reverse transcription polymerase chain reaction (RT-PCR) assays are the most widely used molecular tests for the detection of SARS-CoV-2 and diagnosis of COVID-19 in clinical samples. PCR assays target unique genomic RNA regions to identify SARS-CoV-2 with high sensitivity and specificity. In general, assay development incorporates the whole genome sequences available at design time to be inclusive of all target species and exclusive of near neighbors.

Halo-A Universal Fluorescence Reader Based Threat Agent Detection Platform-A Proof of Concept Study Using SARS-CoV-2 Assays.

Polymerase chain reaction (PCR) remains the gold standard in disease diagnostics due to its extreme sensitivity and specificity. However, PCR tests are expensive and complex, require skilled personnel and specialized equipment to conduct the tests, and have long turnaround times. On the other hand, lateral flow immunoassay-based antigen tests are rapid, relatively inexpensive, and can be performed by untrained personnel at the point of care or even in the home.

Blind Spot: A Braille Patterned Novel Multiplex Lateral Flow Immunoassay Sensor Array for the Detection of Biothreat Agents.

Lateral flow immunoassays (LFIs) are simple, point-of-care diagnostic devices used for detecting biological agents or other analytes of interest in a sample. LFIs are predominantly singleplex assays, interrogating one target analyte at a time. There is a need for multiplex LFI devices, e.

Reorganized Genomic Taxonomy of Enables Design of Robust Environmental PCR Assays for Detection of .

In recent years, an increasing diversity of species has been recognized within the family . Unfortunately, novel isolates are sometimes misnamed in initial publications or multiple sources propose different nomenclature for genetically highly similar isolates. Thus, unstructured and occasionally incorrect information can lead to confusion in the scientific community.

Complete Genome Sequence of Francisella tularensis Live Vaccine Strain NR-28537 (BEI Master Cell Bank).

The genome of live vaccine strain NR-28537 was sequenced by a hybrid approach utilizing an Oxford Nanopore Technologies R9 flow cell and an Illumina MiSeq platform. assembly of the resulting long and short reads produced a single-contig whole-genome sequence.

The State of the Art in Biodefense Related Bacterial Pathogen Detection Using Bacteriophages: How It Started and How It's Going.

Accurate pathogen detection and diagnosis is paramount in clinical success of treating patients. There are two general paradigms in pathogen detection: molecular and immuno-based, and phage-based detection is a third emerging paradigm due to its sensitivity and selectivity. Molecular detection methods look for genetic material specific for a given pathogen in a sample usually by polymerase chain reaction (PCR).

Draft Whole-Genome Sequences of Ciprofloxacin-Resistant Derivatives of a Bacillus anthracis ΔANR Strain Lacking pXO1 and pXO2 Plasmids.

Mutants of an attenuated (ΔANR) strain conferring increasing levels of ciprofloxacin resistance have been described. Here, we report the draft genome sequences of the parent strain (ΔANR pXO1, pXO2) and its derivatives conferring low (step 1; 0.5 μg/ml), medium (step 2; 8 to 16 μg/ml), and high (step 3; 32 to 64 μg/ml) levels of ciprofloxacin resistance.

Exception to the exception rule: synthetic and naturally occurring single chromosome Vibrio cholerae.

The genome of Vibrio cholerae, the etiological agent of cholera, is an exception to the single chromosome rule found in the vast majority of bacteria and has its genome partitioned between two unequally sized chromosomes. This unusual two-chromosome arrangement in V. cholerae has sparked considerable research interest since its discovery.

A Survey of Antimicrobial Resistance Determinants in Category A Select Agents, Exempt Strains, and Near-Neighbor Species.

A dramatic increase in global antimicrobial resistance (AMR) has been well documented. Of particular concern is the dearth of information regarding the spectrum and prevalence of AMR within Category A Select Agents. Here, we performed a survey of horizontally and vertically transferred AMR determinants among Category A agents and their near neighbors.

2018 Ebola virus disease outbreak in Équateur Province, Democratic Republic of the Congo: a retrospective genomic characterisation.

Background: The 2018 Ebola virus disease (EVD) outbreak in Équateur Province, Democratic Republic of the Congo, began on May 8, and was declared over on July 24; it resulted in 54 documented cases and 33 deaths. We did a retrospective genomic characterisation of the outbreak and assessed potential therapeutic agents and vaccine (medical countermeasures).

Methods: We used target-enrichment sequencing to produce Ebola virus genomes from samples obtained in the 2018 Équateur Province outbreak.

Medical countermeasures during the 2018 Ebola virus disease outbreak in the North Kivu and Ituri Provinces of the Democratic Republic of the Congo: a rapid genomic assessment.

Background: The real-time generation of information about pathogen genomes has become a vital goal for transmission analysis and characterisation in rapid outbreak responses. In response to the recently established genomic capacity in the Democratic Republic of the Congo, we explored the real-time generation of genomic information at the start of the 2018 Ebola virus disease (EVD) outbreak in North Kivu Province.

Methods: We used targeted-enrichment sequencing to produce two coding-complete Ebola virus genomes 5 days after declaration of the EVD outbreak in North Kivu.

Functionality of Two Origins of Replication in Strains With a Single Chromosome.

Chromosomal inheritance in bacteria usually entails bidirectional replication of a single chromosome from a single origin into two copies and subsequent partitioning of one copy each into daughter cells upon cell division. However, the human pathogen and other harbor two chromosomes, a large Chr1 and a small Chr2. Chr1 and Chr2 have different origins, an type origin and a P1 plasmid-type origin, respectively, driving the replication of respective chromosomes.

Sensitive detection of live Escherichia coli by bacteriophage amplification-coupled immunoassay on the Luminex® MAGPIX instrument.

Phages are natural predators of bacteria and have been exploited in bacterial detection because of their exquisite specificity to their cognate bacterial hosts. In this study, we present a "proof of concept" bacteriophage amplification-coupled assay as a surrogate for detecting a bacterium present in a sample. The assay entails detection of progeny phage resulting from infection and subsequent growth inside the bacterium present in suspected samples.