Portable Platforms for Molecular-Based Detection of Pathogens in Complex Sample Matrices

Pathogen identification at the point of use is critical in preventing disease transmission and enabling prompt treatment. Current rapid diagnostic tests suffer from high rates of false negatives because they are not capable of detecting the inherently low concentrations of pathogens found in early stages of infection or in environmental reservoirs. The gold standard method for timely pathogen identification is a nucleic acid amplification assay called polymerase chain reaction. Although polymerase chain reaction is extremely sensitive and specific, it requires expensive laboratory equipment and trained personnel to perform the sample preparation, cyclical heating, and amplicon analysis. Isothermal nucleic acid amplification assays are better suited for field use because they operate at a single temperature and are robust to common sample matrix inhibitors. Thus, there is a need to translate isothermal amplification assays to the point of use for rapid and sensitive detection of pathogens in complex samples. Here, I outline an approach to bring laboratory-based sample preparation, assays, and analyses to the point of use via portable platforms. First, I characterize a loop-mediated isothermal amplification assay and combine it with lateral flow immunoassay for simple, colorimetric interpretation of results. Next, I optimize an ambient-temperature reagent storage method to eliminate cold-chain requirements and precision pipetting steps. I then incorporate loop-mediated isothermal amplification, lateral flow immunoassay, and reagent drying into two different integrated paperfluidic platforms and demonstrate their ability to separately detect bacteria and viruses in complex sample matrices. Finally, I couple loop-mediated isothermal amplification with particle diffusometry to optically determine pathogen presence by tracking the Brownian motion of particles added to an amplified sample. The combined loop-mediated isothermal amplification and particle diffusometry method is first characterized on a microscope and then translated to a smartphone-based platform. Each of these portable platforms are broadly applicable because they can be easily modified for identification of other pathogens at the point of use

Feasibility and Acceptability of a Proposed Pharmacy-Based Harm Reduction Intervention to Reduce Opioid Overdose, HIV and Hepatitis C

Background Evidence-based harm reduction intervention components which might benefit pharmacy patients have not been integrated and studied. Objective To investigate the feasibility and acceptability of a proposed pharmacy-based harm reduction intervention to reduce opioid overdose, HIV and hepatitis C called PharmNet. Methods Indiana managing pharmacists were surveyed in 2018 to assess the feasibility and acceptability of an intervention for opioid misuse screening, brief intervention, syringe and naloxone dispensing, and referrals provision. The Consolidated Framework for Implementation Research informed the survey development and analysis. Results The sample included 303 (30.8%) pharmacists; 215 (70.9%) provided detailed written comments. Intervention Characteristics: 83.3% believed PharmNet would benefit patients, and that staff could deliver the intervention with adequate training (70.0%). Inner Setting: While 77.2% believed their pharmacy culture supported practice change, 57.5% of chain pharmacists believed their pharmacies would not have time for PharmNet. Outer Setting: 73.3% believed additional addiction and overdose screening is needed in their community, and pharmacies should offer new services to help reduce opioid overdose and addiction among their patients (79.5%). A vast majority (97.7%) were asked by patients in the past 2 years about syringe related issues; 67.7% were asked about syringes for non-prescription injection drug use. Individuals Involved: While 62.4% believed PharmNet was within pharmacy scope of practice and 90.1% were comfortable consulting about syringe use, pharmacists reported that they had limited control over the implementation environment. Process: 38.0% of pharmacists indicated interest in advising the development of PharmNet. Conclusions An implementation trial of a modified version of PharmNet is likely feasible; yet will be challenged by structural pressures particularly in chain pharmacies. Successful implementation will involve the development of resources and policy components to manage outer and inner setting characteristics and align the intervention to the implementation environment

Strand Displacement Probes Combined with Isothermal Nucleic Acid Amplification for Instrument-Free Detection from Complex Samples

Sensitive and specific detection of pathogens via nucleic acid amplification is currently constrained to laboratory settings and portable equipment with costly fluorescent detectors. Nucleic acid-detecting lateral flow immunoassay strips (LFIAs) offer a low-cost visual transduction strategy at points of need. Unfortunately, these LFIAs frequently detect amplification byproducts that can yield spurious results which can only be deciphered through statistical analysis. We integrated customizable strand displacement probes into standard loop mediated isothermal amplification (LAMP) assays to prevent byproduct capture on commercial LFIAs. We find that combining strand displacement with LAMP (SD-LAMP) yields LFIA test band intensities that can be unequivocally interpreted by human subjects without additional instrumentation, thereby alleviating the need for a portable reader’s analysis. Using SD-LAMP, we capture target amplicons on commercially available LFIAs from as few as 3.5 <i>Vibrio cholerae</i> and 2 750 <i>Escherichia coli</i> bacteria without false positive or false negative interpretation. Moreover, we demonstrate that LFIA capture of SD-LAMP products remain specific even in the presence of complex sample matrixes, providing a significant step toward reliable instrument-free pathogen detection outside of laboratories

Exposure to Alumina Nanoparticles in Female Mice During Pregnancy Induces Neurodevelopmental Toxicity in the Offspring

Alumina nanoparticles (AlNP) have been shown to accumulate in organs and penetrate biological barriers which lead to toxic effects in many organ systems. However, it is not known whether AlNP exposure to female mice during pregnancy can affect the development of the central nervous system or induce neurodevelopmental toxicity in the offspring. The present study aims to examine the effect of AlNP on neurodevelopment and associated underlying mechanism. ICR strain adult female mice were randomly divided into four groups, which were treated with normal saline (control), 10 μm particle size of alumina (bulk-Al), and 50 and 13 nm AlNP during entire pregnancy period. Aluminum contents in the hippocampus of newborns were measured and neurodevelopmental behaviors were tracked in the offspring from birth to 1 month of age. Furthermore, oxidative stress and neurotransmitter levels were measured in the cerebral cortex of the adolescents. Our results showed that aluminum contents in the hippocampus of newborns in AlNP-treated groups were significantly higher than those in bulk-Al and controls. Moreover, the offspring delivered by AlNP-treated female mice displayed stunted neurodevelopmental behaviors. Finally, the offspring of AlNP-treated mice demonstrated significantly increased anxiety-like behavior with impaired learning and memory performance at 1 month of age. The underlying mechanism could be related to increased oxidative stress and decreased neurotransmitter levels in the cerebral cortex. We therefore conclude that AlNP exposure of female mice during pregnancy can induce neurodevelopmental toxicity in offspring

Effect of mild COVID-19 on health-related quality of life

Background: Little is known about the effects of a mild SARS-CoV-2 infection on health-related quality of life. Methods: This prospective observational study of symptomatic adults (18–87 years) who sought outpatient care for an acute respiratory illness, was conducted from 3/30/2020 to 4/30/2021. Participants completed the Short Form Health Survey (SF-12) at enrollment and 6–8 weeks later, to report their physical and mental health function levels as measured by the physical health and mental health composite scores (PHC and MHC, respectively). PHC and MHC scores for COVID-19 cases and non-COVID cases were compared using t-tests. Multivariable regression modeling was used to determine predictors of physical and mental health function at follow-up. Results: Of 2301 enrollees, 426 COVID-19 cases and 547 non-COVID cases completed both surveys. PHC improved significantly from enrollment to follow-up for both COVID-19 cases (5.4 ± 0.41; P < 0.001) and non-COVID cases (3.3 ± 0.32; P < 0.001); whereas MHC improved significantly for COVID-19 cases (1.4 ± 0.51; P < 0.001) and decreased significantly for non-COVID cases (−0.8 ± 0.37; P < 0.05). Adjusting for enrollment PHC, the most important predictors of PHC at follow-up included male sex (β = 1.17; SE = 0.5; P = 0.021), having COVID-19 (β = 1.99; SE = 0.54; P < 0.001); and non-white race (β = −2.01; SE = 0.70; P = 0.004). Adjusting for enrollment MHC, the most important predictors of MHC at follow-up included male sex (β = 1.92; SE = 0.63; P = 0.002) and having COVID-19 (β = 2.42; SE = 0.67; P < 0.001). Conclusion: Both COVID-19 cases and non-COVID cases reported improved physical health function at 6–8 weeks’ convalescence; whereas mental health function improved among COVID-19 cases but declined among non-COVID cases. Both physical and mental health functioning were significantly better among males with COVID-19 than females

An Evolved Mxe GyrA Intein for Enhanced Production of Fusion Proteins

Expressing antibodies as fusions to the non-self-cleaving Mxe GyrA intein enables site-specific, carboxy-terminal chemical modification of the antibodies by expressed protein ligation (EPL). Bacterial antibody-intein fusion protein expression platforms typically yield insoluble inclusion bodies that require refolding to obtain active antibody-intein fusion proteins. Previously, we demonstrated that it was possible to employ yeast surface display to express properly folded single-chain antibody (scFv)-intein fusions, therefore permitting the direct small-scale chemical functionalization of scFvs. Here, directed evolution of the Mxe GyrA intein was performed to improve both the display and secretion levels of scFv-intein fusion proteins from yeast. The engineered intein was shown to increase the yeast display levels of eight different scFvs by up to 3-fold. Additionally, scFv- and green fluorescent protein (GFP)-intein fusion proteins can be secreted from yeast, and while fusion of the scFvs to the wild-type intein resulted in low expression levels, the engineered intein increased scFv-intein production levels by up to 30-fold. The secreted scFv- and GFP-intein fusion proteins retained their respective binding and fluorescent activities, and upon intein release, EPL resulted in carboxy-terminal azide functionalization of the target proteins. The azide-functionalized scFvs and GFP were subsequently employed in a copper-free, strain-promoted click reaction to site-specifically immobilize the proteins on surfaces, and it was demonstrated that the functionalized, immobilized scFvs retained their antigen binding specificity. Taken together, the evolved yeast intein platform provides a robust alternative to bacterial intein expression systems