The future of infectious disease surveillance and outbreak response is trending towards smaller hand-held solutions for point-of-need pathogen detection. Here, samples of Culex cedecei mosquitoes collected in Southern Florida, USA were tested for Venezuelan Equine Encephalitis Virus (VEEV), a previously-weaponized arthropod-borne RNA-virus capable of causing acute and fatal encephalitis in animal and human hosts. A single 20-mosquito pool tested positive for VEEV by quantitative reverse transcription polymerase chain reaction (RT-qPCR) on the Biomeme two3. The virus-positive sample was subjected to unbiased metatranscriptome sequencing on the Oxford Nanopore MinION and shown to contain Everglades Virus (EVEV), an alphavirus in the VEEV serocomplex. Our results demonstrate, for the first time, the use of unbiased sequence-based detection and subtyping of a high-consequence biothreat pathogen directly from an environmental sample using field-forward protocols. The development and validation of methods designed for field-based diagnostic metagenomics and pathogen discovery, such as those suitable for use in mobile “pocket laboratories”, will address a growing demand for public health teams to carry out their mission where it is most urgent: at the point-of-need.

Studies of subsurface microorganisms have yielded few environmentally relevant isolates for laboratory studies. In order to address this lack of cultivated microorganisms, we initiated several enrichments on sediment and underlying basalt samples from North Pond, a sediment basin ringed by basalt outcrops underlying an oligotrophic water-column west of the Mid-Atlantic Ridge at 22°N. In contrast to anoxic enrichments, growth was observed in aerobic, heterotrophic enrichments from sediment of IODP site U1382B at 4 and 68 meters below seafloor (mbsf). These sediment depths, respectively, correspond to the fringes of oxygen penetration from overlying seawater in the top of the sediment column and upward migration of oxygen from oxic seawater from the basalt aquifer below the sediment. Here we report the enrichment, isolation, and initial characterizations of three isolated aerobic heterotrophs from North Pond sediments; an Arthrobacter species from 4 mbsf, and Paracoccus and Pseudomonas species from 68 mbsf. These cultivated bacteria are represented in the amplicon 16S rRNA gene libraries created from whole sediments, albeit at low (up to 2%) relative abundance. We provide genomic evidence from our isolates demonstrating that the Arthrobacter and Pseudomonas isolates have the potential to respire nitrate and oxygen, though dissimilatory nitrate reduction could not be confirmed in laboratory cultures. The cultures from this study represent members of environmentally significant phyla, and allow for further studies into geochemical factors impacting life in the deep subsurface.

Rapid, specific, and sensitive identification of microbial pathogens is critical to infectious disease diagnosis and surveillance. Classical culture-based methods can be applied to a broad range of pathogens but have long turnaround times. Molecular methods, such as PCR, are time-effective but are not comprehensive and may not detect novel strains. Metagenomic shotgun next-generation sequencing (NGS) promises specific identification and characterization of any pathogen (viruses, bacteria, fungi, and protozoa) in a less biased way. Despite its great potential, NGS has yet to be widely adopted by clinical microbiology laboratories due in part to the absence of standardized workflows. Here, we describe a sample-to-answer workflow called PanGIA (Pan-Genomics for Infectious Agents) that includes simplified, standardized wet-lab procedures and data analysis with an easy-to-use bioinformatics tool. PanGIA is an end-to-end, multi-use workflow that can be used for pathogen detection and related applications, such as biosurveillance and biothreat detection. We performed a comprehensive survey and assessment of current, commercially available wet-lab technologies and open-source bioinformatics tools for each workflow component. The workflow includes total nucleic acid extraction from clinical human whole blood and environmental microbial forensic swabs as sample inputs, host nucleic acid depletion, dual DNA and RNA library preparation, shotgun sequencing on an Illumina MiSeq, and sequencing data analysis. The PanGIA workflow can be completed within 24 h and is currently compatible with bacteria and viruses. Here, we present data from the development and application of the clinical and environmental workflows, enabling the specific detection of pathogens associated with bloodstream infections and environmental biosurveillance, without the need for targeted assay development.

Molecular detection of biological agents in the field has traditionally relied on the use of quantitative real-time PCR (qPCR), which now includes commercially available instruments that can be used in the laboratory or field. Adapting this technology for field-forward applications necessitated innovation to minimize size, weight, and power requirements. Rugged, portable instruments, efficient power sources, freeze-dried reagents, data communications, and standard operating procedures for minimally trained users are some examples of limitations that have been overcome to allow qPCR-based data to be generated at the point of need. Despite the high specificity and sensitivity of qPCR, the assays require a priori sequence-based knowledge of the etiological agent to design and produce specific targeted assays with primers and probes. However, in many cases the etiological agent may not be known and pathogen identification must rely on the use of an untargeted screening method. By extracting, preparing, and sequencing all of the genomic material in a particular sample at once, known as metagenomics, a less biased view of the biological entities in that sample can be ascertained. Using metagenomics methods in the field requires the development and optimization of straightforward sample preparation, sequencing, and bioinformatics workflows reminiscent of the challenges faced during the development of field-forward qPCR 15 years ago. To review the state of qPCR and sequencing in the field, we summarized a panel discussion from the 2019 ASM Biothreats Conference. Our discussion focused on the development, evolution, and comparison of molecular methods for biological agents and their utility in the field.

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. Quantitative PCR assays for agents of interest have been successfully used for routine biosurveillance. Recently, there has been increased impetus for adoption of amplicon sequencing (AS) for biosurveillance because it enables discrimination of true positives from near-neighbor false positives, as well as broad, simultaneous detection of many targets in many pathogens in a high-throughput scheme. However, the high sensitivity of AS can lead to false positives. Appropriate controls and workflow reporting can help address these challenges. Objectives: Data reporting standards are critical to data trustworthiness. The standards presented herein aim to provide a framework for method quality assessment in biodetection. Methods: We present a set of standards, Amplicon Sequencing Minimal Information (ASqMI), developed under the auspices of the AOAC INTERNATIONAL Stakeholder Program on Agent Detection Assays for making actionable calls in biosurveillance applications. In addition to the first minimum information guidelines for AS, we provide a controls checklist and scoring scheme to assure AS run quality and assess potential sample contamination. Results: Adoption of the ASqMI guidelines will improve data quality, help track workflow performance, and ultimately provide decision makers confidence to trust the results of this new and powerful technology. Conclusion: AS workflows can provide robust, confident calls for biodetection; however, due diligence in reporting and controls are needed. The ASqMI guideline is the first AS minimum reporting guidance document that also provides the means for end users to evaluate their workflows to improve confidence. Highlights: Standardized reporting guidance for actionable calls is critical to ensuring trustworthy data.