Assessment of a Smartphone-Based Loop-Mediated Isothermal Amplification Assay for Detection of SARS-CoV-2 and Influenza Viruses.

Importance: A critical need exists in low-income and middle-income countries for low-cost, low-tech, yet highly reliable and scalable testing for SARS-CoV-2 virus that is robust against circulating variants. Objective: To assess whether a smartphone-based assay is suitable for SARS-CoV-2 and influenza virus testing without requiring specialized equipment, accessory devices, or custom reagents. Design, Setting, and Participants: This cohort study enrolled 2 subgroups of participants (symptomatic and asymptomatic) at Santa Barbara Cottage Hospital. The symptomatic group consisted of 20 recruited patients who tested positive for SARS-CoV-2 with symptoms; 30 asymptomatic patients were recruited from the same community, through negative admission screening tests for SARS-CoV-2. The smartphone-based real-time loop-mediated isothermal amplification (smaRT-LAMP) was first optimized for analysis of human saliva samples spiked with either SARS-CoV-2 or influenza A or B virus; these results then were compared with those obtained by side-by-side analysis of spiked samples using the Centers for Disease Control and Prevention (CDC) criterion-standard reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) assay. Next, both assays were used to test for SARS-CoV-2 and influenza viruses present in blinded clinical saliva samples obtained from 50 hospitalized patients. Statistical analysis was performed from May to June 2021. Exposures: Testing for SARS-CoV-2 and influenza A and B viruses. Main Outcomes and Measures: SARS-CoV-2 and influenza infection status and quantitative viral load were determined. Results: Among the 50 eligible participants with no prior SARS-CoV-2 infection included in the study, 29 were men. The mean age was 57 years (range, 21 to 93 years). SmaRT-LAMP exhibited 100% concordance (50 of 50 patient samples) with the CDC criterion-standard diagnostic for SARS-CoV-2 sensitivity (20 of 20 positive and 30 of 30 negative) and for quantitative detection of viral load. This platform also met the CDC criterion standard for detection of clinically similar influenza A and B viruses in spiked saliva samples (n = 20), and in saliva samples from hospitalized patients (50 of 50 negative). The smartphone-based LAMP assay was rapid (25 minutes), sensitive (1000 copies/mL), low-cost (<$7/test), and scalable (96 samples/phone). Conclusions and Relevance: In this cohort study of saliva samples from patients, the smartphone-based LAMP assay detected SARS-CoV-2 infection and exhibited concordance with RT-qPCR tests. These findings suggest that this tool could be adapted in response to novel CoV-2 variants and other pathogens with pandemic potential including influenza and may be useful in settings with limited resources.

Smartphone-based pathogen diagnosis in urinary sepsis patients.

BACKGROUND: There is an urgent need for rapid, sensitive, and affordable diagnostics for microbial infections at the point-of-care. Although a number of innovative systems have been reported that transform mobile phones into potential diagnostic tools, the translational challenge to clinical diagnostics remains a significant hurdle to overcome. METHODS: A smartphone-based real-time loop-mediated isothermal amplification (smaRT-LAMP) system was developed for pathogen ID in urinary sepsis patients. The free, custom-built mobile phone app allows the phone to serve as a stand-alone device for quantitative diagnostics, allowing the determination of genome copy-number of bacterial pathogens in real time. FINDINGS: A head-to-head comparative bacterial analysis of urine from sepsis patients revealed that the performance of smaRT-LAMP matched that of clinical diagnostics at the admitting hospital in a fraction of the time (~1 h vs. 18-28 h). Among patients with bacteremic complications of their urinary sepsis, pathogen ID from the urine matched that from the blood - potentially allowing pathogen diagnosis shortly after hospital admission. Additionally, smaRT-LAMP did not exhibit false positives in sepsis patients with clinically negative urine cultures. INTERPRETATION: The smaRT-LAMP system is effective against diverse Gram-negative and -positive pathogens and biological specimens, costs less than $100 US to fabricate (in addition to the smartphone), and is configurable for the simultaneous detection of multiple pathogens. SmaRT-LAMP thus offers the potential to deliver rapid diagnosis and treatment of urinary tract infections and urinary sepsis with a simple test that can be performed at low cost at the point-of-care. FUND: National Institutes of Health, Chan-Zuckerberg Biohub, Bill and Melinda Gates Foundation.

Correcting a Fundamental Flaw in the Paradigm for Antimicrobial Susceptibility Testing.

The emergence and prevalence of antibiotic-resistant bacteria are an increasing cause of death worldwide, resulting in a global 'call to action' to avoid receding into an era lacking effective antibiotics. Despite the urgency, the healthcare industry still relies on a single in vitro bioassay to determine antibiotic efficacy. This assay fails to incorporate environmental factors normally present during host-pathogen interactions in vivo that significantly impact antibiotic efficacy. Here we report that standard antimicrobial susceptibility testing (AST) failed to detect antibiotics that are in fact effective in vivo; and frequently identified antibiotics that were instead ineffective as further confirmed in mouse models of infection and sepsis. Notably, AST performed in media mimicking host environments succeeded in identifying specific antibiotics that were effective in bacterial clearance and host survival, even though these same antibiotics failed in results using standard test media. Similarly, our revised media further identified antibiotics that were ineffective in vivo despite passing the AST standard for clinical use. Supplementation of AST medium with sodium bicarbonate, an abundant in vivo molecule that stimulates global changes in bacterial structure and gene expression, was found to be an important factor improving the predictive value of AST in the assignment of appropriate therapy. These findings have the potential to improve the means by which antibiotics are developed, tested, and prescribed.

Elimination of the cold-chain dependence of a nanoemulsion adjuvanted vaccine against tuberculosis by lyophilization.

Next-generation rationally-designed vaccine adjuvants represent a significant breakthrough to enable development of vaccines against challenging diseases including tuberculosis, HIV, and malaria. New vaccine candidates often require maintenance of a cold-chain process to ensure long-term stability and separate vials to enable bedside mixing of antigen and adjuvant. This presents a significant financial and technological barrier to worldwide implementation of such vaccines. Herein we describe the development and characterization of a tuberculosis vaccine comprised of both antigen and adjuvant components that are stable in a single vial at sustained elevated temperatures. Further this vaccine retains the ability to elicit both antibody and TH1 responses against the vaccine antigen and protect against experimental challenge with Mycobacterium tuberculosis. These results represent a significant breakthrough in the development of vaccine candidates that can be implemented throughout the world without being hampered by the necessity of a continuous cold chain or separate adjuvant and antigen vials.