Species Typing of Nontuberculous Mycobacteria by Use of Deoxyribozyme Sensors.

BACKGROUND: Nontuberculous mycobacteria (NTM) species are a rising threat, especially to patients living with pulmonary comorbidities. Current point-of-care diagnostics fail to adequately identify and differentiate NTM species from Mycobacterium tuberculosis (Mtb). Definitive culture- and molecular-based testing can take weeks to months and requires sending samples out to specialized diagnostic laboratories. METHODS: In this proof-of-concept study, we developed an assay based on PCR amplification of 16S ribosomal RNA (rRNA) rrs genes by using universal mycobacterial primers and interrogation of the amplified fragments with a panel of binary deoxyribozyme (BiDz) sensors to enable species-level identification of NTM (BiDz-NTMST). Each BiDz sensor consists of 2 subunits of an RNA-cleaving deoxyribozyme, which form an active deoxyribozyme catalytic core only in the presence of the complimentary target sequence. The target-activated BiDz catalyzes cleavage of a reporter substrate, thus triggering either fluorescent or colorimetric (visually observed) signal depending on the substrate used. The panel included BiDz sensors for differentiation of 6 clinically relevant NTM species (Mycobacterium abscessus, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium fortuitum, Mycobacterium kansasii, and Mycobacterium gordonae) and Mtb. RESULTS: Using the fluorescent BiDz-NTMST assay, we successfully identified the species of 38 clinical isolates. In addition, a subset of strains was tested with visual BiDz sensors, providing proof-of-concept for species typing of NTM by the naked eye. CONCLUSIONS: The BiDz-NTMST assay is a novel platform for rapid identification of NTM species. This method is highly specific and significantly faster than current tools and is easily adaptable for onsite diagnostic laboratories in hospitals or clinical laboratories.

Development and Optimization of an Unbiased, Metagenomics-Based Pathogen Detection Workflow for Infectious Disease and Biosurveillance Applications.

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.

A randomized, controlled, feasibility study of RD-X19 in subjects with mild-to-moderate COVID-19 in the outpatient setting.

The RD-X19 is an investigational, handheld medical device precisely engineered to emit blue light through the oral cavity to target the oropharynx and surrounding tissues. At doses shown to be noncytotoxic in an in vitro three-dimensional human epithelial tissue model, the monochromatic visible light delivered by RD-X19 results in light-initiated expression of immune stimulating cytokines IL-1α and IL-1ß, with corresponding inhibition of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) replication. A single exposure of 425 nm blue light at 60 J/cm2 led to greater than 99% reductions against all SARS-CoV-2 strains tested in vitro, including the more transmissible (Alpha) and immune evasive (Beta) variants. These preclinical findings along with other studies led to a randomized, double-blind, sham-controlled early feasibility study using the investigational device as a treatment for outpatients with mild to moderate coronavirus disease 2019 (COVID-19). The study enrolled 31 subjects with a positive SARS-CoV-2 antigen test and at least two moderate COVID-19 signs and symptoms at baseline. Subjects were randomized 2:1 (RD-X19: sham) and treated twice daily for 4 days. Efficacy outcome measures included assessments of SARS-CoV-2 saliva viral load and clinical assessments of COVID-19. There were no local application site reactions and no device-related adverse events. At the end of the study (day 8), the mean change in log10 viral load was -3.29 for RD-X19 and -1.81 for sham, demonstrating a treatment benefit of -1.48 logs (95% confidence internal, -2.88 to -0.071, nominal p = 0.040). Among the clinical outcome measures, differences between RD-X19 and sham were also observed, with a 57-h reduction of median time to sustained resolution of COVID-19 signs and symptoms (log rank test, nominal p = 0.044).

Growth hormone-mediated reprogramming of macrophage transcriptome and effector functions.

Macrophages are an important component of the innate immune response. Priming and activation of macrophages is stimulated by cytokines (i.e IFNγ). However, growth hormone (GH) can also stimulate macrophage activation. Based on these observations, the goal of this work was to 1) to compare the transcriptome profile of macrophages activated in vitro with GH and IFNγ, and 2) to assess the impact of GH on key macrophage functional properties like reactive oxygen species (ROS) production and phagosomal proteolysis. To assess the global transcriptional and functional impact of GH on macrophage programming, bone marrow derived macrophages were treated with GH or IFNγ. Our data strongly support a potential link between GH, which wanes with age, and impaired macrophage function. The notable overlap of GH with IFNγ-induced pathways involved in innate immune sensing of pathogens and antimicrobial responses argue for an important role for GH in macrophage priming and maturation. By using functional assays that report on biochemical activities within the lumen of phagosomes, we have also shown that GH alters physiologically relevant processes such as ROS production and proteolysis. These changes could have far reaching impacts on antimicrobial capacity, signaling, and antigen presentation.

Molecular Detection of Biological Agents in the Field: Then and Now.

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.

Molecular drug susceptibility testing and strain typing of tuberculosis by DNA hybridization.

In Mycobacterium tuberculosis (Mtb) the detection of single nucleotide polymorphisms (SNPs) is of high importance both for diagnostics, since drug resistance is primarily caused by the acquisition of SNPs in multiple drug targets, and for epidemiological studies in which strain typing is performed by SNP identification. To provide the necessary coverage of clinically relevant resistance profiles and strain types, nucleic acid-based measurement techniques must be able to detect a large number of potential SNPs. Since the Mtb problem is pressing in many resource-poor countries, requiring low-cost point-of-care biosensors, this is a non-trivial technological challenge. This paper presents a proof-of-concept in which we chose simple DNA-DNA hybridization as a sensing principle since this can be transferred to existing low-cost hardware platforms, and we pushed the multiplex boundaries of it. With a custom designed probe set and a physicochemical-driven data analysis it was possible to simultaneously detect the presence of SNPs associated with first- and second-line drug resistance and Mtb strain typing. We have demonstrated its use for the identification of drug resistance and strain type from a panel of phylogenetically diverse clinical strains. Furthermore, reliable detection of the presence of a minority population (<5%) of drug-resistant Mtb was possible.

Stromal cells expressing elevated VCAM-1 enhance survival of B lineage tumor cells.

In the current study we generated murine bone marrow stromal cells that constitutively express human VCAM-1. These stromal cell lines allow investigation of the contribution of VCAM-1 initiated signaling to tumor cell survival. Co-culture of ALL cell lines with stromal cells engineered to over express VCAM-1 enhanced survival of leukemic cells in a PI-3 kinase-dependent manner, compared to co-culture with parental stromal cells expressing only endogenous VCAM-1. These observations suggest that modulation of stromal cell VCAM-1 by specific chemotherapeutic drugs may have utility in decreasing residual disease. In addition, these novel lines provide an in vitro model in which other tumor types that interact with stromal cells in the bone marrow microenvironment may be evaluated to determine the contribution of VCAM-1 initiated signaling to modulation of treatment response.