Microfluidic Diffusional Sizing (MDS) Measurements of Secretory Neutralizing Antibody Affinity Against SARS-CoV-2.

SARS-CoV-2 has rampantly spread around the globe and continues to cause unprecedented loss through ongoing waves of (re)infection. Increasing our understanding of the protection against infection with SARS-CoV-2 is critical to ending the pandemic. Serological assays have been widely used to assess immune responses, but secretory antibodies, the essential first line of defense, have been studied to only a limited extent. Of particular interest and importance are neutralizing antibodies, which block the binding of the spike protein of SARS-CoV-2 to the human receptor angiotensin-converting enzyme-2 (ACE2) and thus are essential for immune defense. Here, we employed Microfluidic Diffusional Sizing (MDS), an immobilization-free technology, to characterize neutralizing antibody affinity to SARS-CoV-2 spike receptor-binding domain (RBD) and spike trimer in saliva. Affinity measurement was obtained through a contrived sample and buffer using recombinant SARS-CoV-2 RBD and monoclonal antibody. Limited saliva samples demonstrated that MDS applies to saliva neutralizing antibody measurement. The ability to disrupt a complex of ACE2-Fc and spike trimer is shown. Using a quantitative assay on the patient sample, we determined the affinity and binding site concentration of the neutralizing antibodies.

Guide to assembling a successful K99/R00 application.

The National Institutes of Health's (NIH) K99/R00 Pathway to Independence Award offers promising postdoctoral researchers and clinician-scientists an opportunity to receive research support at both the mentored and the independent levels with the goal of facilitating a timely transition to a tenure-track faculty position. This transitional program has been generally successful, with most K99/R00 awardees successfully securing R01-equivalent funding by the end of the R00 period. However, often highly promising proposals fail because of poor grantsmanship. This overview provides guidance from the perspective of long-standing members of the National Heart, Lung, and Blood Institute's Mentored Transition to Independence study section for the purpose of helping mentors and trainees regarding how best to assemble competitive K99/R00 applications.

Repurposing a SARS-CoV-2 surveillance program for infectious respiratory diseases in a university setting.

Standard multiplex RT-qPCR diagnostic tests use nasopharyngeal swabs to simultaneously detect a variety of infections, but commercially available kits can be expensive and have limited throughput. Previously, we clinically validated a saliva-based RT-qPCR diagnostic test for SARS-CoV-2 to provide low-cost testing with high throughput and low turnaround time on a university campus. Here, we developed a respiratory diagnostic panel to detect SARS-CoV-2, influenza A and B within a single saliva sample. When compared to clinical results, our assay demonstrated 93.5% accuracy for influenza A samples (43/46 concordant results) with no effect on SARS-CoV-2 accuracy or limit of detection. In addition, our assay can detect simulated coinfections at varying virus concentrations generated from synthetic RNA controls. We also confirmed the stability of influenza A in saliva at room temperature for up to 5 days. The cost of the assay is lower than standard nasopharyngeal swab respiratory panel tests as saliva collection does not require specialized swabs or trained clinical personnel. By repurposing the lab infrastructure developed for the COVID-19 pandemic, our multiplex assay can be used to provide expanded access to respiratory disease diagnostics, especially for community, school, or university testing applications where saliva testing was effectively utilized during the COVID-19 pandemic.

A capacitive laser-induced graphene based aptasensor for SARS-CoV-2 detection in human saliva.

SARS-CoV-2 virus induced CoVID-19 pandemic has accelerated the development of diagnostic tools. Devices integrated with electrochemical biosensors may be an interesting alternative to respond to the high demand for testing, particularly in contexts where access to standard detection technologies is lacking. Aptamers as recognition elements are useful due to their stability, specificity, and sensitivity to binding target molecules. We have developed a non-invasive electrochemical aptamer-based biosensor targeting SARS-CoV-2 in human saliva. The aptamer is expected to detect the Spike protein of SARS-CoV-2 wildtype and its variants. Laser-induced graphene (LIG) electrodes coated with platinum nanoparticles were biofunctionalized with a biotin-tagged aptamer. Electrochemical Impedance Spectroscopy (EIS) for BA.1 sensing was conducted in sodium chloride/sodium bicarbonate solution supplemented with pooled saliva. To estimate sensing performance, the aptasensor was tested with contrived samples of UV-attenuated virions from 10 to 10,000 copies/ml. Selectivity was assessed by exposing the aptasensor to non-targeted viruses (hCoV-OC43, Influenza A, and RSV-A). EIS data outputs were further used to select a suitable response variable and cutoff frequency. Capacitance increases in response to the gradual loading of the attenuated BA.1. The aptasensor was sensitive and specific for BA.1 at a lower viral load (10-100 copies/ml) and was capable of discriminating between negative and positive contrived samples (with strain specificity against other viruses: OC43, Influenza A, and RSV-A). The aptasensor detected SARS-CoV-2 with an estimated LOD of 1790 copies/ml in contrived samples. In human clinical samples, the aptasensor presents an accuracy of 72%, with 75% of positive percent of agreement and 67% of negative percent of agreement. Our results show that the aptasensor is a promising candidate to detect SARS-CoV-2 during early stages of infection when virion concentrations are low, which may be useful for preventing the asymptomatic spread of CoVID-19.

Optimization of tumor-treating field therapy for triple-negative breast cancer cells in vitro via frequency modulation.

PURPOSE: Currently, tumor-treating field (TTField) therapy utilizes a single "optimal" frequency of electric fields to achieve maximal cell death in a targeted population of cells. However, because of differences in cell size, shape, and ploidy during mitosis, optimal electric field characteristics for universal maximal cell death may not exist. This study investigated the anti-mitotic effects of modulating electric field frequency as opposed to utilizing uniform electric fields. METHODS: We developed and validated a custom device that delivers a wide variety of electric field and treatment parameters including frequency modulation. We investigated the efficacy of frequency modulating tumor-treating fields on triple-negative breast cancer cells compared to human breast epithelial cells. RESULTS: We show that frequency-modulated (FM) TTFields are as selective at treating triple-negative breast cancer (TNBC) as uniform TTFields while having a greater efficacy for combating TNBC cell growth. TTField treatment at a mean frequency of 150 kHz with a frequency range of ± 10 kHz induced apoptosis in a greater number of TNBC cells after 24 h as compared to unmodulated treatment which led to further decreased cell viability after 48 h. Furthermore, all TNBC cells died after 72 h of FM treatment while cells that received unmodulated treatment were able to recover to cell number equivalent to the control. CONCLUSION: TTFields were highly efficacious against TNBC growth, FM TTFields showed minimal effects on epithelial cells similar to unmodulated treatment.

Identification and diagnosis of long COVID-19: A scoping review.

Long COVID-19 (LC-19) is a condition that has affected a high percentage of the population that recovered from the initial disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). LC-19 diagnosis is currently poorly defined because of its variable, multisystem, episodic symptoms, and lack of uniformity in the critical time points associated with the disease. Considering the number of cases, workers' compromised efficiency or inability to return to their duties can affect organizations and impact economies. LC-19 represents a significant burden on multiple levels and effectively reduces quality of life. These factors necessitate the establishment of firm parameters of diagnoses to provide a foundation for ongoing and future studies of clinical characteristics, epidemiology, risk factors, and therapy. In this scoping review, we conducted a literature search across multiple publication sites to identify papers of interest regarding the diagnosis of LC-19. We identified 225 records of interest and categorized them into seven categories. Based on our findings, there are only 11 original papers that outline the diagnostic process in detail with little overlap. This scoping review highlights the lack of consensus regarding the definition and, thereby, the LC-19 diagnosis processes. Due to no clear directive and considering the many unknowns surrounding the natural history of the disease and further recovery/sequelae from COVID-19, continued discussion and agreement on a definition/diagnosis will help future research and management of these patients.

Efficacy and selectivity of tumor-treating field therapy for triple-negative breast cancer cells via in-house delivery device.

PURPOSE: Triple-negative breast cancer continues to be one of the leading causes of death in women, making up 7% of all cancer deaths. Tumor-treating electric fields are low-energy, low-frequency oscillating electric fields that induce an anti-proliferative effect on mitotic cells in glioblastoma multiforme, non-small cell lung cancer, and ovarian cancer. Little is known about effects of tumor-treating fields on triple-negative breast cancer and known research for tumor-treating fields only utilizes low (< 3 V/cm) electric field intensities. METHODS: We have developed an in-house field delivery device capable of high levels of customization to explore a much wider variety of electric field and treatment parameters. Furthermore, we investigated the selectivity of tumor-treating field treatment between triple-negative breast cancer and human breast epithelial cells. RESULTS: Tumor-treating fields show greatest efficacy against triple-negative breast cancer cell lines between 1 and 3 V/cm electric field intensities while having little effect on epithelial cells. CONCLUSION: These results provide a clear therapeutic window for tumor-treating field delivery to triple-negative breast cancer.

Covid-19 vaccine effectiveness against general SARS-CoV-2 infection from the omicron variant: A retrospective cohort study.

We aim to estimate the effectiveness of 2-dose and 3-dose mRNA vaccination (BNT162b2 and mRNA-1273) against general Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection (asymptomatic or symptomatic) caused by the omicron BA.1 variant. This propensity-score matched retrospective cohort study takes place in a large public university undergoing weekly Coronavirus Disease 2019 (Covid-19) testing in South Carolina, USA. The population consists of 24,145 university students and employees undergoing weekly Covid-19 testing between January 3rd and January 31st, 2022. The analytic sample was constructed via propensity score matching on vaccination status: unvaccinated, completion of 2-dose mRNA series (BNT162b2 or mRNA-1273) within the previous 5 months, and receipt of mRNA booster dose (BNT162b2 or mRNA-1273) within the previous 5 months. The resulting analytic sample consists of 1,944 university students (mean [SD] age, 19.64 [1.42] years, 66.4% female, 81.3% non-Hispanic White) and 658 university employees (mean [SD] age, 43.05 [12.22] years, 64.7% female, 83.3% non-Hispanic White). Booster protection against any SARS-CoV-2 infection was 66.4% among employees (95% CI: 46.1-79.0%; P<.001) and 45.4% among students (95% CI: 30.0-57.4%; P<.001). Compared to the 2-dose mRNA series, estimated increase in protection from the booster dose was 40.8% among employees (P=.024) and 37.7% among students (P=.001). We did not have enough evidence to conclude a statistically significant protective effect of the 2-dose mRNA vaccination series, nor did we have enough evidence to conclude that protection waned in the 5-month period after receipt of the 2nd or 3rd mRNA dose. Furthermore, we did not find evidence that protection varied by manufacturer. We conclude that in adults 18-65 years of age, Covid-19 mRNA booster doses offer moderate protection against general SARS-CoV-2 infection caused by the omicron variant and provide a substantial increase in protection relative to the 2-dose mRNA vaccination series.

Simultaneous assessment of radial and axial myocyte mechanics by combining atomic force microscopy and carbon fibre techniques.

Cardiomyocytes sense and shape their mechanical environment, contributing to its dynamics by their passive and active mechanical properties. While axial forces generated by contracting cardiomyocytes have been amply investigated, the corresponding radial mechanics remain poorly characterized. Our aim is to simultaneously monitor passive and active forces, both axially and radially, in cardiomyocytes freshly isolated from adult mouse ventricles. To do so, we combine a carbon fibre (CF) set-up with a custom-made atomic force microscope (AFM). CF allows us to apply stretch and to record passive and active forces in the axial direction. The AFM, modified for frontal access to fit in CF, is used to characterize radial cell mechanics. We show that stretch increases the radial elastic modulus of cardiomyocytes. We further find that during contraction, cardiomyocytes generate radial forces that are reduced, but not abolished, when cells are forced to contract near isometrically. Radial forces may contribute to ventricular wall thickening during contraction, together with the dynamic re-orientation of cells and sheetlets in the myocardium. This new approach for characterizing cell mechanics allows one to obtain a more detailed picture of the balance of axial and radial mechanics in cardiomyocytes at rest, during stretch, and during contraction. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.

Development of a Biosensor Based on Angiotensin-Converting Enzyme II for Severe Acute Respiratory Syndrome Coronavirus 2 Detection in Human Saliva.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the novel coronavirus responsible for COVID-19. Infection in humans requires angiotensin-converting enzyme II (hACE2) as the point of entry for SARS-CoV-2. PCR testing is generally definitive but expensive, although it is highly sensitive and accurate. Biosensor-based monitoring could be a low-cost, accurate, and non-invasive approach to improve testing capacity. We develop a capacitive hACE2 biosensor for intact SARS-CoV-2 detection in saliva. Laser-induced graphene (LIG) electrodes were modified with platinum nanoparticles. The quality control of LIG electrodes was performed using cyclic voltammetry. Truncated hACE2 was used as a biorecognition element and attached to the electrode surface by streptavidin-biotin coupling. Biolayer interferometry was used for qualitative interaction screening of hACE2 with UV-attenuated virions. Electrochemical impedance spectroscopy (EIS) was used for signal transduction. Truncated hACE2 binds wild-type SARS-CoV-2 and its variants with greater avidity than human coronavirus (common cold virus). The limit of detection (LoD) is estimated to be 2,960 copies/ml. The detection process usually takes less than 30 min. The strength of these features makes the hACE2 biosensor a potentially low-cost approach for screening SARS-CoV-2 in non-clinical settings with high demand for rapid testing (for example, schools and airports).

Effectiveness and protection duration of Covid-19 vaccines and previous infection against any SARS-CoV-2 infection in young adults.

Data on effectiveness and protection duration of Covid-19 vaccines and previous infection against general SARS-CoV-2 infection in general populations are limited. Here we evaluate protection from Covid-19 vaccination (primary series) and previous infection in 21,261 university students undergoing repeated surveillance testing between 8/8/2021-12/04/2021, during which B.1.617 (delta) was the dominant SARS-CoV-2 variant. Estimated mRNA-1273, BNT162b2, and AD26.COV2.S effectiveness against any SARS-CoV-2 infection is 75.4% (95% CI: 70.5-79.5), 65.7% (95% CI: 61.1-69.8), and 42.8% (95% CI: 26.1-55.8), respectively. Among previously infected individuals, protection is 72.9% when unvaccinated (95% CI: 66.1-78.4) and increased by 22.1% with full vaccination (95% CI: 15.8-28.7). Statistically significant decline in protection is observed for mRNA-1273 (P < .001), BNT162b2 (P < .001), but not Ad26.CoV2.S (P = 0.40) or previous infection (P = 0.12). mRNA vaccine protection dropped 29.7% (95% CI: 17.9-41.6) six months post- vaccination, from 83.2% to 53.5%. We conclude that the 2-dose mRNA vaccine series initially offers strong protection against general SARS-CoV-2 infection caused by the delta variant in young adults, but protection substantially decreases over time. These findings indicate that vaccinated individuals may still contribute to community spread. While previous SARS-CoV-2 infection consistently provides moderately strong protection against repeat infection from delta, vaccination yields a substantial increase in protection.

Identifying SARS-CoV-2 Variants of Concern through Saliva-Based RT-qPCR by Targeting Recurrent Mutation Sites.

SARS-CoV-2 variants of concern (VOCs) continue to pose a public health threat which necessitates a real-time monitoring strategy to complement whole genome sequencing. Thus, we investigated the efficacy of competitive probe RT-qPCR assays for six mutation sites identified in SARS-CoV-2 VOCs and, after validating the assays with synthetic RNA, performed these assays on positive saliva samples. When compared with whole genome sequence results, the SΔ69-70 and ORF1aΔ3675-3677 assays demonstrated 93.60 and 68.00% accuracy, respectively. The SNP assays (K417T, E484K, E484Q, L452R) demonstrated 99.20, 96.40, 99.60, and 96.80% accuracies, respectively. Lastly, we screened 345 positive saliva samples from 7 to 22 December 2021 using Omicron-specific mutation assays and were able to quickly identify rapid spread of Omicron in Upstate South Carolina. Our workflow demonstrates a novel approach for low-cost, real-time population screening of VOCs. IMPORTANCE SARS-CoV-2 variants of concern and their many sublineages can be characterized by mutations present within their genetic sequences. These mutations can provide selective advantages such as increased transmissibility and antibody evasion, which influences public health recommendations such as mask mandates, quarantine requirements, and treatment regimens. Our RT-qPCR workflow allows for strain identification of SARS-CoV-2 positive saliva samples by targeting common mutation sites shared between variants of concern and detecting single nucleotides present at the targeted location. This differential diagnostic system can quickly and effectively identify a wide array of SARS-CoV-2 strains, which can provide more informed public health surveillance strategies in the future.

SARS-CoV-2 variants of concern Alpha and Delta show increased viral load in saliva.

BACKGROUND: Higher viral loads in SARS-CoV-2 infections may be linked to more rapid spread of emerging variants of concern (VOC). Rapid detection and isolation of cases with highest viral loads, even in pre- or asymptomatic individuals, is essential for the mitigation of community outbreaks. METHODS AND FINDINGS: In this study, we analyze Ct values from 1297 SARS-CoV-2 positive patient saliva samples collected at the Clemson University testing lab in upstate South Carolina. Samples were identified as positive using RT-qPCR, and clade information was determined via whole genome sequencing at nearby commercial labs. We also obtained patient-reported information on symptoms and exposures at the time of testing. The lowest Ct values were observed among those infected with Delta (median: 22.61, IQR: 16.72-28.51), followed by Alpha (23.93, 18.36-28.49), Gamma (24.74, 18.84-30.64), and the more historic clade 20G (25.21, 20.50-29.916). There was a statistically significant difference in Ct value between Delta and all other clades (all p.adj<0.01), as well as between Alpha and 20G (p.adj<0.05). Additionally, pre- or asymptomatic patients (n = 1093) showed the same statistical differences between Delta and all other clades (all p.adj<0.01); however, symptomatic patients (n = 167) did not show any significant differences between clades. Our weekly testing strategy ensures that cases are caught earlier in the infection cycle, often before symptoms are present, reducing this sample size in our population. CONCLUSIONS: COVID-19 variants Alpha and Delta have substantially higher viral loads in saliva compared to more historic clades. This trend is especially observed in individuals who are pre- or asymptomatic, which provides evidence supporting higher transmissibility and more rapid spread of emerging variants. Understanding the viral load of variants spreading within a community can inform public policy and clinical decision making.

Implementation of a Rural Community Diagnostic Testing Strategy for SARS-CoV-2 in Upstate South Carolina.

By developing a partnership amongst a public university lab, local city government officials and community healthcare providers, we established a drive-through COVID-19 testing site aiming to improve access to SARS-CoV-2 testing in rural Upstate South Carolina. We collected information on symptoms and known exposures of individuals seeking testing to determine the number of pre- or asymptomatic individuals. We completed 71,102 SARS-CoV-2 tests in the community between December 2020-December 2021 and reported 91.49% of results within 24 h. We successfully identified 5,244 positive tests; 73.36% of these tests originated from individuals who did not report symptoms. Finally, we identified high transmission levels during two major surges and compared test positivity rates of the local and regional communities. Importantly, the local community had significantly lower test positivity rates than the regional community throughout 2021 (p < 0.001). While both communities reached peak case load and test positivity near the same time, the local community returned to moderate transmission as indicated by positivity 4 weeks before the regional community. Our university lab facilitated easy testing with fast turnaround times, which encouraged voluntary testing and helped identify a large number of non-symptomatic cases. Finding the balance of simplicity, accessibility, and community trust was vital to the success of our widespread community testing program for SARS-CoV-2.

Identifying SARS-CoV-2 Variants of Concern through saliva-based RT-qPCR by targeting recurrent mutation sites.

SARS-CoV-2 variants of concern (VOCs) continue to pose a public health threat which necessitates a real-time monitoring strategy to compliment whole genome sequencing. Thus, we investigated the efficacy of competitive probe RT-qPCR assays for six mutation sites identified in SARS-CoV-2 VOCs and, after validating the assays with synthetic RNA, performed these assays on positive saliva samples. When compared with whole genome sequence results, the SΔ69-70 and ORF1aΔ3675-3677 assays demonstrated 93.60% and 68.00% accuracy, respectively. The SNP assays (K417T, E484K, E484Q, L452R) demonstrated 99.20%, 96.40%, 99.60%, and 96.80% accuracies, respectively. Lastly, we screened 345 positive saliva samples from December 7-22, 2021 using Omicron-specific mutation assays and were able to quickly identify rapid spread of Omicron in Upstate South Carolina. Our workflow demonstrates a novel approach for low-cost, real-time population screening of VOCs. Importance: SARS-CoV-2 variants of concern and their many sublineages can be characterized by mutations present within their genetic sequences. These mutations can provide selective advantages such as increased transmissibility and antibody evasion, which influences public health recommendations such as mask mandates, quarantine requirements, and treatment regimens. Our real-time RT-qPCR workflow allows for strain identification of SARS-CoV-2 positive saliva samples by targeting common mutation sites shared between VOCs and detecting single nucleotides present at the targeted location. This differential diagnostic system can quickly and effectively identify a wide array of SARS-CoV-2 strains, which can provide more informed public health surveillance strategies in the future.

Virtual article collection on infectious diseases.

Specifically for COVID-19, we have had several recent articles on SARS-CoV-2. Sohail and Nutini reported on models working to predict the incubation period for SARS-CoV-2 and disease progression. Güler et al. wrote a review of the biophysical and biochemical properties of SARS-CoV-2 which highlighted how the virus's molecular structure allows it to interact and infect cells. These structures are also potential targets for diagnostic and treatment strategies. Lalitha Guruprasad's review on how the various human coronavirus spike proteins interact with human cell proteins and carbohydrate receptors provides further insight on coronavirus-cell interactions as well, and reviews successfully repurposed drugs to combat coronavirus-based diseases.

SARS-CoV-2 variants of concern Alpha and Delta show increased viral load in saliva.

BACKGROUND: Higher viral loads in SARS-CoV-2 infections may be linked to more rapid spread of emerging variants of concern (VOC). Rapid detection and isolation of cases with highest viral loads, even in pre- or asymptomatic individuals, is essential for the mitigation of community outbreaks. METHODS AND FINDINGS: In this study, we analyze Ct values from 1297 SARS-CoV-2 positive patient saliva samples collected at the Clemson University testing lab in upstate South Carolina. Samples were identified as positive using RT-qPCR, and clade information was determined via whole genome sequencing at nearby commercial labs. We also obtained patient-reported information on symptoms and exposures at the time of testing. The lowest Ct values were observed among those infected with Delta (median: 22.61, IQR: 16.72-28.51), followed by Alpha (23.93, 18.36-28.49), Gamma (24.74, 18.84-30.64), and the more historic clade 20G (25.21, 20.50-29.916). There was a statistically significant difference in Ct value between Delta and all other clades (all p.adj<0.01), as well as between Alpha and 20G (p.adj<0.05). Additionally, pre- or asymptomatic patients (n=1093) showed the same statistical differences between Delta and all other clades (all p.adj<0.01); however, symptomatic patients (n=167) did not show any significant differences between clades. Our weekly testing strategy ensures that cases are caught earlier in the infection cycle, often before symptoms are present, reducing this sample size in our population. CONCLUSIONS: COVID-19 variants Alpha and Delta have substantially higher viral loads in saliva compared to more historic clades. This trend is especially observed in individuals who are pre- or asymptomatic, which provides evidence supporting higher transmissibility and more rapid spread of emerging variants. Understanding the viral load of variants spreading within a community can inform public policy and clinical decision making.

Efficient SARS-CoV-2 Quantitative Reverse Transcriptase PCR Saliva Diagnostic Strategy utilizing Open-Source Pipetting Robots.

The emergence of the recent SARS-CoV-2 global health crisis introduced key challenges for epidemiological research and clinical testing. Characterized by a high rate of transmission and low mortality, the COVID-19 pandemic necessitated accurate and efficient diagnostic testing, particularly in closed populations such as residential universities. Initial availability of nucleic acid testing, like nasopharyngeal swabs, was limited due to supply chain pressure which also delayed reporting of test results. Saliva-based reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) testing has shown to be comparable in sensitivity and specificity to other testing methods, and saliva collection is less physically invasive to participants. Consequently, we developed a multiplex RT-qPCR diagnostic assay for population surveillance of Clemson University and the surrounding community. The assay utilized open-source liquid handling robots and thermocyclers instead of complex clinical automation systems to optimize workflow and system flexibility. Automation of saliva-based RT-qPCR enables rapid and accurate detection of a wide range of viral RNA concentrations for both large- and small-scale testing demands. The average turnaround for the automated system was < 9 h for 95% of samples and < 24 h for 99% of samples. The cost for a single test was $2.80 when all reagents were purchased in bulk quantities.