Chagas disease is an inflammatory parasitic infection caused by Trypanosoma cruzi (T. cruzi). Early diagnosis is crucial in guiding treatment and slowing disease progression; however, current diagnostic methods have insufficient detection limits and often require skilled technicians. Molecular tests, especially isothermal nucleic acid assays, are advantageous due to their excellent sensitivity, specificity, speed, and simplicity. Here, we optimized a colorimetric loop-mediated isothermal amplification (LAMP) assay for T. cruzi. We can detect as few as 2 genomic copies/reaction using three different T. cruzi strains. We examined selectivity using other parasitic protozoans and successfully detected T. cruzi DNA extracted from parasites in human whole blood down to 1.2 parasite equivalents/reaction. We also performed a blinded study using canine blood samples and established a 100% sensitivity, specificity, and accuracy for the colorimetric LAMP assay. Finally, we used a heated 3D printer bed and an insulated thermos cup to demonstrate that the LAMP incubation step could be performed with accessible, low-cost materials. Altogether, we have developed a high-performing assay for T. cruzi with a simple colorimetric output that would be ideal for rapid, low-cost screening at the point of use.

A portable microfluidic platform uses a novel programmable magnetic actuation mechanism to process samples for sensitive detection of inflammatory biomarkers.

Testing is pivotal for early identification of disease and subsequent infection control. Pathogens' nucleic acid sequence can change due to naturally-occurring genetic drift or intentional modification. Because of the reliance on molecular assays for human, animal, and plant disease diagnosis, we must understand how nucleotide mutations affect test accuracy. Primers designed against original lineages of a pathogen may be less efficient at detecting variants with genetic changes in priming regions. Here, we made single- and multi-point mutations in priming regions of a model SARS-CoV-2 template that was used as input for a loop-mediated isothermal amplification (LAMP) assay. We found that many of the modifications impacted assay sensitivity, amplification speed, or both. Further research exploring mutations at every position in each of the eight priming regions should be conducted to evaluate trends and determine generalizability. Nucleotide substitutions and deletions can slow or completely inhibit nucleic acid amplification compared to unmodified control samples.

Many ground beetle species (Coleoptera: Carabidae) prey on noctuid larvae and pupae. Therefore, agricultural practices that maintain or even en-hance carabid populations have the potential to reduce noctuid pest populations through predation. One such pest is the fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), a migratory pest of row, turf, and vegetable crops. Before migrating in spring from southern Florida and southern Texas, it feeds and develops on cover crops such as field corn and sorghum-sudangrass and expands its populations. Here we sampled the species and density of carabid populations that were active in cover crops with differing capacity to support fall armyworm development. Three cover crop species, sorghum-sudangrass, cowpea, and sunn hemp, were previously studied for their effects on fall armyworm populations, with sunn hemp showing high incompatibility with fall armyworm development. The cover crops were grown in 3 locations in north and north-central Florida and pitfall traps were used to compare numbers of carabid beetles caught in different cover crop treatments. Almost 2,000 predatory and omnivorous carabid beetles were collected. Three species in particular, Calosoma sayi Dejean, Tetracha carolina (L.), and Cicindela punctulata Olivier, are known predators of S. frugiperda in the laboratory and may aid in the reduction of populations in the field. There were no differences in beetle numbers among cover crop plants. In another trial, more beetles were collected in plots of a popular sunn hemp cultivar developed in the southeastern U.S., 'AU Golden', than in plots of another germplasm line, Tillage SunnTM. Further research should determine if the predatory species found in this study prey on S. frugiperda in the field and if this added mortality helps reduce crop damage.

IntroductionThe SARS-CoV-2 pandemic, and the subsequent limitations on standard diagnostics, has vastly expanded the user base of Reverse Transcription Loop-mediated isothermal Amplification (RT-LAMP) in fundamental research and development. RT-LAMP has also penetrated commercial markets, with emergency use authorizations for clinical diagnosis.Areas coveredThis review discusses the role of RT-LAMP within the context of other technologies like RT-qPCR and rapid antigen tests, progress in sample preparation strategies to enable simplified workflow for RT-LAMP directly from clinical specimens, new challenges with primer and assay design for the evolving pandemic, prominent detection modalities including colorimetric and CRISPR-mediated methods, and translational research and commercial development of RT-LAMP for clinical applications.Expert opinionRT-LAMP occupies a middle ground between RT-qPCR and rapid antigen tests. The simplicity approaches that of rapid antigen tests, making it suitable for point-of-care use, but the sensitivity nears that of RT-qPCR. RT-LAMP still lags RT-qPCR in fundamental understanding of the mechanism, and the interplay between sample preparation and assay performance. Industry is now beginning to address issues around scalability and usability, which could finally enable LAMP and RT-LAMP to find future widespread application as a diagnostic for other conditions, including other pathogens with pandemic potential.

We present a field-deployable microfluidic immunoassay device in response to the need for sensitive, quantitative, and highthroughput protein detection at point-of-need.The portable microfluidic system facilitates eight magnetic bead-based sandwich immunoassays from raw samples in 45 minutes.An innovative bead actuation strategy was incorporated into the system to automate multiple sample process steps with minimal user intervention.The device is capable of quantitative and sensitive protein analysis with a 10 pg/ml detection limit from interleukin 6-spiked human serum samples.We envision the reported device offering ultrasensitive point-of-care immunoassay tests for timely and accurate clinical diagnosis.

Here, we describe the isolation of 18 unique anti SARS-CoV-2 human single-chain antibodies from an antibody library derived from healthy donors. The selection used a combination of phage and yeast display technologies and included counter-selection strategies meant to direct the selection of the receptor-binding motif (RBM) of SARS-CoV-2 spike protein's receptor binding domain (RBD2). Selected antibodies were characterized in various formats including IgG, using flow cytometry, ELISA, high throughput SPR, and fluorescence microscopy. We report antibodies' RBD2 recognition specificity, binding affinity, and epitope diversity, as well as ability to block RBD2 binding to the human receptor angiotensin-converting enzyme 2 (ACE2) and to neutralize authentic SARS-CoV-2 virus infection in vitro. We present evidence supporting that: 1) most of our antibodies (16 out of 18) selectively recognize RBD2; 2) the best performing 8 antibodies target eight different epitopes of RBD2; 3) one of the pairs tested in sandwich assays detects RBD2 with sub-picomolar sensitivity; and 4) two antibody pairs inhibit SARS-CoV-2 infection at low nanomolar half neutralization titers. Based on these results, we conclude that our antibodies have high potential for therapeutic and diagnostic applications. Importantly, our results indicate that readily available non immune (naïve) antibody libraries obtained from healthy donors can be used to select high-quality monoclonal antibodies, bypassing the need for blood of infected patients, and offering a widely accessible and low-cost alternative to more sophisticated and expensive antibody selection approaches (e.g. single B cell analysis and natural evolution in humanized mice).

The fall armyworm (FAW; Spodoptera frugiperda) is one of the major agricultural pest insects. FAW is native to the Americas, and its invasion was first reported in West Africa in 2016. Then it quickly spread through Africa, Asia, and Oceania, becoming one of the main threats to corn production. We analyzed whole genome sequences of 177 FAW individuals from 12 locations on four continents to infer evolutionary processes of invasion. Principal component analysis from the TPI gene and whole genome sequences shows that invasive FAW populations originated from the corn strain. Ancestry coefficient and phylogenetic analyses from the nuclear genome indicate that invasive populations are derived from a single ancestry, distinct from native populations, while the mitochondrial phylogenetic tree supports the hypothesis of multiple introductions. Adaptive evolution specific to invasive populations was observed in detoxification, chemosensory, and digestion genes. We concluded that extant invasive FAW populations originated from the corn strain with potential contributions of adaptive evolution.

Introduction: Over the past decade, loop-mediated isothermal amplification (LAMP) technology has played an important role in molecular diagnostics. Amongst numerous nucleic acid amplification assays, LAMP stands out in terms of sample-to-answer time, sensitivity, specificity, cost, robustness, and accessibility, making it ideal for field-deployable diagnostics in resource-limited regions. Areas covered: In this review, we outline the front-end LAMP design practices for point-of-care (POC) applications, including sample handling and various signal readout methodologies. Next, we explore existing LAMP technologies that have been validated with clinical samples in the field. We summarize recent work that utilizes reverse transcription (RT) LAMP to rapidly detect SARS-CoV-2 as an alternative to standard PCR protocols. Finally, we describe challenges in translating LAMP from the benchtop to the field and opportunities for future LAMP assay development and performance reporting. Expert opinion: Despite the popularity of LAMP in the academic research community and a recent surge in interest in LAMP due to the COVID-19 pandemic, there are numerous areas for improvement in the fundamental understanding of LAMP, which are needed to elevate the field of LAMP assay development and characterization.

Eleven Labs within the US Department of Energy (DOE), National Virtual Biotechnology Laboratory (NVBL), came together as a team to address significant R&D gaps in COVID-19 testing. Beginning in March 2020, the NVBL COVID Testing Team developed an R&D agenda, worked with DOE and other agencies to set priorities, and collaborated to deliver timely results. Priority was given to quick implementation as well as development of novel capabilities for immediate and evolving pandemic needs without placing additional burden on operational performers. Priority elements capitalized on DOE National Laboratory strengths and expertise. The Team delivered: testing and evaluation that enabled decisions on testing options, forwardleaning approaches to prepare for future scale-up needs, and models and experiments that supported prioritization of diagnostic and therapeutic candidates.

The fall armyworm (FAW), Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), is a highly polyphagous plant pest that can severely impact yields of several agricultural crops. Understanding the economic impact and management thresholds for FAW across a variety of crop commodities is crucial for effective management. Evaluating the peer-reviewed and grey literature, we compiled global data on: (1) yield losses reported as a result of FAW infestations, (2) the relationship between FAW pressure/density and reported yield loss, and (3) current known economic injury levels, economic thresholds and action thresholds. We identified 71 references that reported yield losses from FAW infestation, with a total of 888 separate yield loss entries. The majority of research quantifying yield losses and the relationship between pest pressure and yield has focused on maize, sorghum, and cotton, with some evidence for sweet corn, bermudagrass, and rice. Yield loss varied between management strategies, with genetically modified and/or insecticide treated crops typically retaining higher yields. Most studies investigating the relationship between FAW density and yield loss across different crops have focused on early and mid FAW larval instars and on vegetative through to reproductive plant growth stages, with minimal research on both late larval instars and on plant seedlings. Economic thresholds were not reported in the literature. The reporting of economic injury levels and action thresholds varied significantly both between and within crops, highlighting the need for a standardised approach when measuring FAW pressures or densities that elicit management responses.

BackgroundThe fall armyworm (Spodoptera frugiperda (J.E. Smith)) is a highly polyphagous agricultural pest with long-distance migratory behavior threatening food security worldwide. This pest has a host range of >80 plant species, but two host strains are recognized based on their association with corn (C-strain) or rice and smaller grasses (R-strain). The population genomics of the United States (USA) fall armyworm remains poorly characterized to date despite its agricultural threat.ResultsIn this study, the population structure and genetic diversity in 55S. frugiperda samples from Argentina, Brazil, Kenya, Puerto Rico and USA were surveyed to further our understanding of whole genome nuclear diversity. Comparisons at the genomic level suggest a panmictic S. frugiperda population, with only a minor reduction in gene flow between the two overwintering populations in the continental USA, also corresponding to distinct host strains at the mitochondrial level. Two maternal lines were detected from analysis of mitochondrial genomes. We found members from the Eastern Hemisphere interspersed within both continental USA overwintering subpopulations, suggesting multiple individuals were likely introduced to Africa.ConclusionsOur research is the largest diverse collection of United States S. frugiperda whole genome sequences characterized to date, covering eight continental states and a USA territory (Puerto Rico). The genomic resources presented provide foundational information to understand gene flow at the whole genome level among S. frugiperda populations. Based on the genomic similarities found between host strains and laboratory vs. field samples, our findings validate the experimental use of laboratory strains and the host strain differentiation based on mitochondria and sex-linked genetic markers extends to minor genome wide differences with some exceptions showing mixture between host strains is likely occurring in field populations.

Respiratory viral infections are considered a major public health threat, and breath metabolomics can provide new ways to detect and understand how specific viruses affect the human pulmonary system. In this pilot study, we characterized the metabolic composition of human breath for an early diagnosis and differentiation of influenza viral infection, as well as other types of upper respiratory viral infections. We first studied the non-specific effects of planned seasonal influenza vaccines on breath metabolites in healthy subjects after receiving the immunization. We then investigated changes in breath content from hospitalized patients with flu-like symptoms and confirmed upper respiratory viral infection. The exhaled breath was sampled using a custom-made breath condenser, and exhaled breath condensate (EBC) samples were analysed using liquid chromatography coupled to quadruplole-time-of-flight mass spectrometer (LC-qTOF). All metabolomic data was analysed using both targeted and untargeted approaches to detect specific known biomarkers from inflammatory and oxidative stress biomarkers, as well as new molecules associated with specific infections. We were able to find clear differences between breath samples collected before and after flu vaccine administration, together with potential biomarkers that are related to inflammatory processes and oxidative stress. Moreover, we were also able to discriminate samples from patients with flu-related symptoms that were diagnosed with confirmatory respiratory viral panels (RVPs). RVP positive and negative differences were identified, as well as differences between specific viruses defined. These results provide very promising information for the further study of the effect of influenza A and other viruses in human systems by using a simple and non-invasive specimen like breath.

Staphylococcus aureus is a major human pathogen of the skin. The global burden of diabetes is high, with S. aureus being a major complication of diabetic wound infections. We investigated how the diabetic environment influences S. aureus skin infection and observed an increased susceptibility to infection in mouse models of both type I and type II diabetes. A dual gene expression approach was taken to investigate transcriptional alterations in both the host and bacterium after infection. While analysis of the host response revealed only minor changes between infected control and diabetic mice, we observed that S. aureus isolated from diabetic mice had significant increases in the levels of genes associated with translation and posttranslational modification and chaperones and reductions in the levels of genes associated with amino acid transport and metabolism. One family of genes upregulated in S. aureus isolated from diabetic lesions encoded the Clp proteases, associated with the misfolded protein response. The Clp proteases were found to be partially glucose regulated as well as influencing the hemolytic activity of S. aureus Strains lacking the Clp proteases ClpX, ClpC, and ClpP were significantly attenuated in our animal model of skin infection, with significant reductions observed in dermonecrosis and bacterial burden. In particular, mutations in clpP and clpX were significantly attenuated and remained attenuated in both normal and diabetic mice. Our data suggest that the diabetic environment also causes changes to occur in invading pathogens, and one of these virulence determinants is the Clp protease system.

Are lawn caterpillars doing your mowing for you? Young caterpillars, or larvae, injure turfgrass by chewing notches along the edge of the leaves. This creates a ragged appearance (Figure 1) that may be hard to notice at first. Mature caterpillars eat a lot before they pupate and consume patches of turfgrass down to the crown. Because the turf looks scalped so quickly, people think that the damage occurs “overnight.” Several caterpillar species can be turfgrass pests, including the tropical sod webworm, the fall armyworm, and the striped grass looper. This document is ENY-352 (IN608), one of a series of the Department of Entomology and Nematology, UF/IFAS Extension. Original publication date: April 2006.

Stenotrophomonas maltophilia is a Gram-negative opportunistic pathogen that can infect the lungs of people with cystic fibrosis (CF). The highly viscous mucus in the CF lung, expectorated as sputum, serves as the primary nutrient source for microbes colonizing this site and induces virulence-associated phenotypes and gene expression in several CF pathogens. Here, we characterized the transcriptional responses of three S. maltophilia strains during exposure to synthetic CF sputum medium (SCFM2) to gain insight into how this organism interacts with the host in the CF lung. These efforts led to the identification of 881 transcripts differentially expressed by all three strains, many of which reflect the metabolic pathways used by S. maltophilia in sputum, as well as altered stress responses. The latter correlated with increased resistance to peroxide exposure after pregrowth in SCFM2 for two of the strains. We also compared the SCFM2 transcriptomes of two S. maltophilia CF isolates to that of the acute infection strain, S. maltophilia K279a, allowing us to identify CF isolate-specific signatures in differential gene expression. The expression of genes from the accessory genomes was also differentially altered in response to SCFM2. Finally, a number of biofilm-associated genes were differentially induced in SCFM2, particularly in K279a, which corresponded to increased aggregation and bio-film formation in this strain relative to both CF strains. Collectively, this work details the response of S. maltophilia to an environment that mimics important aspects of the CF lung, identifying potential survival strategies and metabolic pathways used by S. maltophilia during infections. IMPORTANCE Stenotrophomonas maltophilia is an important infecting bacterium in the airways of people with cystic fibrosis (CF). However, compared to the other CF pathogens, S. maltophilia has been relatively understudied. The significance of our research is to provide insight into the global transcriptomic changes of S. maltophilia in response to a medium that was designed to mimic important aspects of the CF lung. This study elucidates the overall metabolic changes that occur when S. maltophilia encounters the CF lung and generates a road map of candidate genes to test using in vitro and in vivo models of CF.

The recent discovery of fall armyworm (Spodoptera frugiperda, J.E. Smith) in Africa presents a significant threat to that continent’s food security. The species exhibits several traits in the Western Hemisphere that if transferred to Africa would significantly complicate control efforts. These include a broad host range, long-distance migratory behavior, and resistance to multiple pesticides that varies by regional population. Therefore, determining which fall armyworm subpopulations are present in Africa could have important implications for risk assessments and mitigation efforts. The current study is an extension of earlier surveys that together combine the collections from 11 nations to produce the first genetic description of fall armyworm populations spanning the sub-Saharan region. Comparisons of haplotype frequencies indicate significant differences between geographically distant populations. The haplotype profile from all locations continue to identify Florida and the Caribbean regions as the most likely Western Hemisphere origins of the African infestations. The current data confirm the uncertainty of fall armyworm strain identification in Africa by genetic methods, with the possibility discussed that the African infestation may represent a novel interstrain hybrid population of potentially uncertain behavioral characteristics.