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As the COVID-19 pandemic progresses, there is an increasing need for rapid, accessible assays for SARS-CoV-2 detection. We present a clinical evaluation and real-world implementation of the INDICAID COVID-19 rapid antigen test (INDICAID rapid test). A multisite clinical evaluation of the INDICAID rapid test using prospectively collected nasal (bilateral anterior) swab samples from symptomatic subjects was performed. The INDICAID rapid test demonstrated a positive percent agreement (PPA) and negative percent agreement (NPA) of 85.3% (95% confidence interval [95% CI], 75.6% to 91.6%) and 94.9% (95% CI, 91.6% to 96.9%), respectively, compared to laboratory-based reverse transcriptase PCR (RT-PCR) using nasal specimens. The INDICAID rapid test was then implemented at COVID-19 outbreak screening centers in Hong Kong as part of a testing algorithm (termed “dual-track”) to screen asymptomatic individuals for prioritization for confirmatory RT-PCR testing. In one approach, preliminary positive INDICAID rapid test results triggered expedited processing for laboratory-based RT-PCR, reducing the average time to confirmatory result from 10.85 h to 7.0 h. In a second approach, preliminary positive results triggered subsequent testing with an onsite rapid RT-PCR, reducing the average time to confirmatory result to 0.84 h. In 22,994 asymptomatic patients, the INDICAID rapid test demonstrated a PPA of 84.2% (95% CI, 69.6% to 92.6%) and an NPA of 99.9% (95% CI, 99.9% to 100%) compared to laboratory-based RT-PCR using combined nasal/oropharyngeal specimens. The INDICAID rapid test has excellent performance compared to laboratory-based RT-PCR testing and, when used in tandem with RT-PCR, reduces the time to confirmatory positive result. IMPORTANCE Laboratory-based RT-PCR, the current gold standard for COVID-19 testing, can require a turnaround time of 24 to 48 h from sample collection to result. The delayed time to result limits the effectiveness of centralized RT-PCR testing to reduce transmission and stem potential outbreaks. To address this, we conducted a thorough evaluation of the INDICAID COVID-19 rapid antigen test, a 20-minute rapid antigen test, in both symptomatic and asymptomatic populations. The INDICAID rapid test demonstrated high sensitivity and specificity with RT-PCR as the comparator method. A dual-track testing algorithm was also evaluated utilizing the INDICAID rapid test to screen for preliminary positive patients, whose samples were then prioritized for RT-PCR testing. The dual-track method demonstrated significant improvements in expediting the reporting of positive RT-PCR test results compared to standard RT-PCR testing without prioritization, offering an improved strategy for community testing and controlling SARS-CoV-2 outbreaks.

Low yields of extracted cell-free DNA (cfDNA) from plasma limit continued development of liquid biopsy in cancer, especially in early-stage cancer diagnostics and cancer screening applications. We investigate a novel liquid-phase-based DNA isolation method that utilizes aqueous two-phase systems to purify and concentrate circulating cfDNA. The PHASIFY MAX and PHASIFY ENRICH kits were compared to a commonly employed solid-phase extraction method on their ability to extract cfDNA from a set of 91 frozen plasma samples from cancer patients. Droplet digital PCR (ddPCR) was used as the downstream diagnostic to detect mutant copies. Compared to the QIAamp Circulating Nucleic Acid (QCNA) kit, the PHASIFY MAX method demonstrated 60% increase in DNA yield and 171% increase in mutant copy recovery, and the PHASIFY ENRICH kit demonstrated a 35% decrease in DNA yield with a 153% increase in mutant copy recovery. A follow-up study with PHASIFY ENRICH resulted in the positive conversion of 9 out of 47 plasma samples previously determined negative with QCNA extraction (all with known positive tissue genotyping). Our results indicate that this novel extraction technique offers higher cfDNA recovery resulting in better sensitivity for detection of cfDNA mutations compared to a commonly used solid-phase extraction method.

Background: Low yields of extracted cell-free DNA (cfDNA) from plasma limit further development of liquid biopsies for early-stage cancer diagnostics and cancer screening applications. Conventional nucleic acid extraction methods have limitations in capturing low levels of cfDNA and risk capturing high levels of high molecular weight contaminating genomic DNA. We developed a liquid-phase based DNA extraction method (PHASIFY™ ENRICH; Phase Scientific International Ltd., Kwun Tong, Hong Kong) that leverages aqueous two-phase systems (ATPS) to purify, concentrate, and enrich for cfDNA to improve mutant signal detection. Methods: The PHASIFY ATPS consists of unique formulations that are optimized to drive cfDNA into one liquid phase and plasma contaminants into the other, achieving simultaneous target isolation and concentration. In a previous study using the PHASIFY technique, we demonstrated improved cfDNA and mutant copy yield over solid-phase methods. We further developed an additional size-selection feature (PHASIFY ENRICH) to remove high molecular weight genomic DNA (>500 bp) and to reduce signal noise for molecular diagnostics. We compared cfDNA recovery and mutation detection from plasma samples extracted with the industry standard QIAamp Circulating Nucleic Acid kit (QCNA; Qiagen, Hilden, Germany) and PHASIFY ENRICH cfDNA extraction kit. According to the respective kit protocols, the cfDNA was extracted from plasma from patients with advanced cancers, including those known to shed low amounts of cfDNA into circulation (e.g., low grade serious ovarian, appendiceal, thyroid, and pancreatic cancer), with known tissue mutation status. Total DNA recovery was determined with the Quant-iT PicoGreen dsDNA Assay Kit. Mutation status was determined with probe-based Droplet Digital PCR (ddPCR; Bio-Rad, Hercules, CA). In a follow up study, we tested a similar cohort of plasma samples that were previously tested negative for cfDNA mutation status when using QCNA cfDNA extraction, despite known positive mutation status from corresponding tumor tissue genotyping. Results: In the head-to-head study against QCNA, 57 plasma samples from 23 unique patients with diverse advanced cancers with known tissue mutation status were used for cfDNA extraction. The PHASIFY ENRICH method resulted in a 153% increase in median mutant copy recovery compared to QCNA (P = 0.01 Wilcoxon Signed-Rank). In the follow up study, 47 plasma samples from 31 patients with diverse advanced cancers and prior cfDNA negative mutation status with tissue positive mutation status were used for extraction. Of the 47samples extracted by PHASIFY ENRICH, 9 samples resulted in a conversion to positive cfDNA mutation status compared to no mutant signals when previously using QCNA. Conclusion: Our results indicate that PHASIFY ENRICH provides improved cfDNA mutation detection by increasing cfDNA recovery and reducing contaminating genomic DNA background. Citation Format: Filip Janku, Helen Huang, David Y. Pereira, Masae Kobayashi, Steven G. Call, Kristen T. Woodbury, Felix Chao, Daniel R. Marshak, Ricky Y. Chiu. Enhancing cell-free DNA mutation detection with novel liquid-phase extraction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 575.

Background: Low yields of extracted cell-free DNA (cfDNA) from plasma limit further development of liquid biopsies especially in early-stage cancer diagnostics and cancer screening applications. We developed a novel liquid-liquid based DNA isolation method (PHASIFY™ MAX; Phase Scientific International Ltd., Kwun Tong, Hong Kong) that leverages aqueous two-phase systems (ATPS) to purify and concentrate circulating cfDNA, which can result in increase of cfDNA recovery and mutant signal detection. Methods: The PHASIFY™ ATPS consists of unique formulations of polymer and salt components that can intrinsically separate into two distinct phases. The formula can be optimized to drive cfDNA into one phase and contaminants from plasma into the other, achieving simultaneous target isolation and concentration. We compared cfDNA recovery and mutation detection from plasma samples extracted with the industry standard QIAamp Circulating Nucleic Acid kit (QCNA; Qiagen, Hilden, Germany) and PHASIFY™ MAX cfDNA extraction kit. According to the respective kit protocols, the cfDNA was extracted from 1mL of plasma from patients with advanced cancers, including those known to shed low amounts of cfDNA into circulation (e.g., thyroid, pancreatic), with known tissue BRAF, KRAS, or NRAS mutation status. Total DNA recovery was determined with the Quant-iT PicoGreen dsDNA Assay Kit. Mutation status was determined with probe-based (BRAF, KRAS, NRAS) Droplet Digital PCR (ddPCR; Bio-Rad, Hercules, CA). Results: Plasma samples from 89 patients with diverse advanced cancers (colorectal, N=36; ovarian, N=18; lung N=7; thyroid, N=6; pancreatic, N=6; others, N=16) with known BRAF, KRAS or NRAS mutation status were used for cfDNA extraction with standard QCNA and PHASIFY™ MAX kits. On average, PHASIFY™ MAX demonstrated a 288% increase in total cfDNA recovery compared to the QCNA kit (p-value Conclusion: Our early results indicate that the PHASIFY™ MAX extraction technique offers higher cfDNA recovery resulting in higher sensitivity of cfDNA mutation detection compared to the industry standard. Citation Format: Filip Janku, Helen J. Huang, David Y. Pereira, Masae Kobayashi, Chung Hei Chiu, Steven G. Call, Kristen T. Woodbury, Felix Chao, Daniel R. Marshak, Ricky Y. Chiu. Enhancing cell-free DNA and mutant copy recovery from plasma with novel liquid-liquid extraction [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1979.

Availability of a rapid, accurate, and reliable point-of-care (POC) device for detection of infectious agents and pandemic pathogens, such as swine-origin influenza A (H1N1) virus, is crucial for effective patient management and outbreak prevention. Due to its ease of use, rapid processing, and minimal power and laboratory equipment requirements, the lateral-flow (immuno)assay (LFA) has gained much attention in recent years as a possible solution. However, since the sensitivity of LFA has been shown to be inferior to that of the gold standards of pathogen detection, namely cell culture and real-time PCR, LFA remains an ineffective POC assay for preventing pandemic outbreaks. A practical solution for increasing the sensitivity of LFA is to concentrate the target agent in a solution prior to the detection step. In this study, an aqueous two-phase micellar system comprised of the nonionic surfactant Triton X-114 was investigated for concentrating a model virus, namely bacteriophage M13 (M13), prior to LFA. The volume ratio of the two coexisting micellar phases was manipulated to concentrate M13 in the top, micelle-poor phase. The concentration step effectively improved the M13 detection limit of the assay by tenfold from 5 × 108 plaque forming units (pfu)/mL to 5 × 107 pfu/mL. In the future, the volume ratio can be further manipulated to yield a greater concentration of a target virus and further decrease the detection limits of the LFA. Figure A schematic representation of concentrating viruses with an aqueous two-phase micellar system containing Triton X-114 surfactant prior to the detection of the virus through the lateral-flow immunoassay Electronic supplementary material The online version of this article (doi:10.1007/s00216-010-4213-7) contains supplementary material, which is available to authorized users.

Glioblastoma multiforme (GBM) is the most common and lethal primary brain tumor with median survival of only 12 to 15 months under the current standard of care. To both increase tumor specificity and decrease nonspecific side effects, recent experimental strategies in the treatment of GBM have focused on targeting cell surface receptors, including the transferrin (Tf) receptor, that are overexpressed in many cancers. A major limitation of Tf-based therapeutics is the short association of Tf within the cell to deliver its payload. We previously developed two mutant Tf molecules, K206E/R632A Tf and K206E/K534A Tf, in which iron is locked into each of the two homologous lobes. Relative to wild-type Tf, we showed enhanced delivery of diphtheria toxin (DT) from these mutants to a monolayer culture of HeLa cells. Here, we extend the application of our Tf mutants to the treatment of GBM. In vitro treatment of Tf mutants to a monolayer culture of glioma cells showed enhanced cellular association as well as enhanced delivery of conjugated DT. Treatment of GBM xenografts with mutant Tf-conjugated DT resulted in pronounced regression in vivo, indicating their potential use as drug carriers. Cancer Res; 70(11); 4520–7. ©2010 AACR.