Simplified Paper Format for Detecting HIV Drug Resistance in Clinical Specimens by Oligonucleotide Ligation

Human immunodeficiency virus (HIV) is a chronic infection that can be managed by antiretroviral treatment (ART). However, periods of suboptimal viral suppression during lifelong ART can select for HIV drug resistant (DR) variants. Transmission of drug resistant virus can lessen or abrogate ART efficacy. Therefore, testing of individuals for drug resistance prior to initiation of treatment is recommended to ensure effective ART. Sensitive and inexpensive HIV genotyping methods are needed in low-resource settings where most HIV infections occur. The oligonucleotide ligation assay (OLA) is a sensitive point mutation assay for detection of drug resistance mutations in HIV pol. The current OLA involves four main steps from sample to analysis: (1) lysis and/or nucleic acid extraction, (2) amplification of HIV RNA or DNA, (3) ligation of oligonucleotide probes designed to detect single nucleotide mutations that confer HIV drug resistance, and (4) analysis via oligonucleotide surface capture, denaturation, and detection (CDD). The relative complexity of these steps has limited its adoption in resource-limited laboratories. Here we describe a simplification of the 2.5-hour plate-format CDD to a 45-minute paper-format CDD that eliminates the need for a plate reader. Analysis of mutations at four HIV-1 DR codons (K103N, Y181C, M184V, and G190A) in 26 blood specimens showed a strong correlation of the ratios of mutant signal to total signal between the paper CDD and the plate CDD. The assay described makes the OLA easier to perform in low resource laboratories

Development of platforms for detection of single nucleotide polymorphisms: applications on pathogens, genetic diseases, and beyond

Thesis (Ph.D.)--University of Washington, 2021HIV was once a pandemic, but development of drugs has allowed people to live long healthy lives despite infection with HIV. Unfortunately, HIV has developed resistance to many drugs and threatens to become a pandemic again. In 2019, 1 out of 4 infected people had HIV with mutations that render first-line drugs ineffective. The situation was much worse among infants born to HIV-infected mothers, of whom 3 out of 4 had drug-resistant HIV. In response to this pressing issue, the WHO recommends that countries switch their first-line treatments to dolutegravir-based regimens. For countries that continue to use the old first-line drugs, WHO recommends testing individuals for HIV drug resistance (HIVDR). HIVDR test results can guide clinicians to select the most effective treatment. Unfortunately, HIVDR tests are typically only available in advanced centralized laboratories, and a whole country our continent may be served by a single WHO-certified testing site. The primary aim of this thesis was to address this urgent unmet need improving speed, cost, and affordability of an existing HIVDR testing method. Single nucleotide polymorphisms (SNPs) are proven biomarkers for HIVDR. Chapter 1 Introduction provides the background on SNPs, their relationship with HIVDR, the magnitude of the HIVDR problem, and existing technologies to detect these HIVDR SNPs. Materials related to this chapter are included in the following publications.1) Horacio A Duarte, Nuttada Panpradist, Ingrid A Beck, Barry Lutz, James Lai, Ruth M Kanthula, Rami Kantor, Anubhav Tripathi, Shanmugam Saravanan, Iain J MacLeod, Michael H Chung, Guoqing Zhang, Chunfu Yang, Lisa M Frenkel. “Current status of point-of-care testing for human immunodeficiency virus drug resistance” J Infect Dis. 2017;216(suppl_9): S824-S828. PMID: 29040621. DOI: 10.1093/infdis/jix413. 2) Nuttada Panpradist, James J Lai. “Point-of-care diagnostics” 2016. Biomaterials Nanoarchitectonics. 2016. DOI: 10.1016/B978-0-323-37127-8.00009-1. 3) Ross S. Milne, Ingrid A. Beck, Molly Levine, Isaac So, Nina Anderson, Wenjie Deng, Thomas R. Sibley, Nuttada Panpradist, James Kingoo, Catherine Kiptinness, Nelly Yatich, James N Kiarie, Samah R Sakr, Michael H Chung, Lisa M Frenkel. “Low-frequency pretreatment HIV drug-resistance effects on 2-year outcome of first-line efavirenz-based antiretroviral therapy” submitted to AIDS. Chapters 2 – 5 describe how we simplified SNP detection based on the oligonucleotide ligation assay (OLA) pioneered by the Frenkel lab nearly 20 years ago. The OLA consists of four main modules: sample preparation, amplification, ligase detection reaction (LDR), and detection of the ligated probes, which indicates the presence or absence of a particular SNP. Specifically, Chapter 2 Nucleic acid preparation describes the significance of using DNA vs. RNA and how to prepare them for the HIVDR test. We developed non-proprietary RNA extraction kits and evaluated them on clinical specimens. Chapter 3 Paper-based detection for HIVDR showcases three generations of OLA detection that rely on the principles of paper microfluidics, nucleic acid thermodynamics, protein chemistries, and multi-target binding interactions. Herein I also discuss the comparisons of human visual classification and software analysis to improve signal-noise discrimination via linear discriminant analysis. Chapter 4 Ready-to-use dry reagents details the strategies for preparing lyophilized, ready-to-use reagents in different platforms, selecting appropriate excipients, characterization methods, additional considerations for complex molecular assays, and the potential use of Arrhenius’s equation in accelerated long-term storage study. Combinations of this know-how in Chapter 2 – Chapter 4 are the basis of developing “OLA-Simple 1.0 kits for HIVDR test” in Chapter 5 OLA-Simple kit for HIVDR. First, the OLA-Simple kit was made “universal” and used to validate an archived specimen panel of 168 specimens (672 SNPs tested) containing globally represented subtypes from Thailand (AE and B), South Africa (C), Peru (B), and Kenya (A, D, and AD). Next, the OLA-Simple kit was tailored for subtype-B specific HIV and tested on circulating variants in Mexico City in 60 specimens (300 SNPs tested); at that time, the country was not a part of the dolutegravir patent pool and would likely have benefitted from the OLA-Simple kits. Last, an interactive Aquarium software guide was developed through a new collaboration with the Klavins lab to enable the use of OLA-Simple kits with minimal or no training. This software guide, added to the OLA-Simple kits, resulted in successful demonstrations in the USA (41 participants) and Kenya (12 participants). My contributions in these areas led to the following publications. The complete lists are available in Appendix 7 – Appendix 9.  PUBLICATIONS 1) Nuttada Panpradist, Ingrid A Beck, Michael H Chung, James N Kiarie, Lisa M Frenkel, Barry R Lutz. “Simplified paper format for detecting HIV drug resistance in clinical specimens by oligonucleotide ligation.” PLoS One. 2016. DOI: 10.1371/journal.pone.0145962 2) Nuttada Panpradist, Ingrid A Beck, Justin Vrana, Nikki Higa, David McIntyre, Parker S Ruth, Isaac So, Enos C Kline, Ruth Kanthula, Annie Wong-On-Wing, Jonathan Lim, Daisy Ko, Ross Milne, Theresa Rossouw, Ute D Feucht, Michael Chung, Gonzague Jourdain, Nicole Ngo-Giang-Huong, Laddawan Laomanit, Jaime Soria, James Lai, Eric D Klavins, Lisa M Frenkel, Barry R Lutz. “OLA-Simple: A software-guided HIV-1 drug resistance test for low-resource laboratories”. EBiomedicine. 2019. DOI: 10.1016/j.ebiom.2019.11.002 3) Nuttada Panpradist*, Ingrid A Beck*, Parker S Ruth, Santiago Avila-Rios, Claudia Garcia-Morales, Maribel Soto-Nava, Daniela Tapia-Trejo, Margarita Matias-Florentino, Hector E Paz-Juarez, Silvia Del Arenal-Sanchez, Gustovo Reyes-Teran, Barry R Lutz, Lisa M Frenkel. “Near point-of-care, point-mutation test to detect drug resistance in HIV-1: A validation study in a Mexican cohort.” AIDS. 2020. PMID:32205723 DOI: 10.1097/QAD.0000000000002524 * Authors with equal contributions. 4) Justin D Vrana*, Nuttada Panpradist*, Nikki Higa, Daisy Ko, Parker Ruth, Ruth Kanthula, James J Lai, Yaoyu Yang, Samar Rafie Sakr, Bhavna Chohan, Michael H Chung, Lisa M Frenkel, Barry R Lutz, Eric Klavins, Ingrid A Beck. “Implementation of an interactive mobile application to pilot a rapid assay to detect HIV drug resistance mutations in Kenya.” medRxiv. DOI: 10.1101/2021.05.06.21256654 *Authors with equal contributions. 5) Gaurav Gulati*, Nuttada Panpradist*, Samuel W A Stewart, Amy K Oreskovic, Santiago Avila, Gustavo Reyes-Terán, Peter D Han, Lea M Starita, Ingrid Beck, Lisa M Frenkel, Barry R Lutz, James J Lai. “Inexpensive workflow to enable simultaneous screening for Covid-19 and monitoring HIV viral load” medRXiv. PMID: 34462759 DOI: 10.1101/2021.08.18.21256786. * Authors with equal contributions. PATENT APPLICATIONS1) PCT Application WO 2019/222716 filed on 05/17/2019 Entitled: “Systems and Methods for ligation. Chapter 6 Strategies to improve amplification and ligation for HIVDR proposes an effective workflow for a viral load test and reflexed HIVDR test, eliminating redundancies in sample collection and extraction and speeding up test turnaround time while reducing costs. I discuss our efforts to increase speed further, reduce complexity, and integrate the amplification and ligation module in the OLA-Simple. Five hours of sample-to-results time in OLA-Simple 1.0 could be reduced to as low as 1 hour. We used different techniques to enable isothermal molecular reactions, such as thermostable helicase-dependent amplification, recombinase polymerase amplification, and loop-mediated amplification. Chapter 7 Fluorescence-based detection of LDR products describes novel strategies to chemically integrate all steps into a single reaction tube using fluorescent-based detection approaches via reverse-molecular beacons robust to sequence polymorphisms in HIV. Chapter 8 Integration of HIVDR OLA into a more simplified device highlights different methods we applied to physically integrate OLA into a device, such as a 1-dimensional sequential delivery, a magnetic valve, a pull tab valve, and a wax-based encapsulation and release system. This work was reported in the following conference proceedings and manuscripts: CONFERENCE PROCEEDINGS1) N Panpradist, EC Kline, IA Beck, AK Oreskovic, I Hull, M Purfield, S Avila-Ríos, C García-Morales, G Reyes- Terán, LM Frenkel, BR Lutz. “Rapid semi-quantitative viral load assay and reflex drug resistance test for management of HIV antiretroviral therapy.” 28th International Workshop on HIV Drug Resistance & Treatment Strategies. 2019. Johannesburg, South Africa. (Poster Presentation) 2) N Panpradist, D McIntyre, A Wong-On-Wing, A Sriram, I Beck, L Frenkel, B Lutz. “Development of a Near Equipment-free, Self-Contained HIV Drug Resistance Test.” Biomedical Engineering Society Conference. 2017. Phoenix, Arizona, USA. (Oral Presentation) 3) N Panpradist, A Wong-On-Wing, I Beck, J Lai, L Frenkel, B Lutz. “Progress towards Development of a Novel Isothermal Ligation Reaction for HIV Drug Resistance Testing.” XXV International Workshop on HIV Drug Resistance. 2016. Boston, MA. (Poster Presentation) 4) N Panpradist, N Higa, A Wong-On-Wing, I Andrews, B Atkinson, I Beck, L Frenkel, B Lutz. “Novel Platform for Simple Lab-based Oligonucleotide Ligation Assay for Detection of HIV Drug Resistance in sub-Saharan Africans.” XXV International Workshop on HIV Drug Resistance. Boston, MA. 2016 (Poster Presentation). SUBMITTED MANUSCRIPT1) Ian T. Hull, Enos C. Kline, Gaurav K. Gulati, Jack Henry Kotnik, Nuttada Panpradist, Kamal G. Shah, Qin Wang, Lisa Frenkel, James Lai, Joanne Stekler, and Barry R. Lutz. “Isothermal Amplification with a Target-Mimicking Internal Control and Quantitative Lateral Flow Readout for Rapid HIV Viral Load Testing in Low-Resource Settings” accepted to Analytical Chemistry. MANUSCRIPT IN PREPARATION:1) Nuttada Panpradist*, Amy Oreskovic*, Enos Kline, Ian Hull, Parker Ruth, Ingrid A Beck, Santiago Avila-Rios, Michael Chung, Ute Feucht, Lisa Frenkel, Gonzhang Jourdain, Enos Kline, Jonathan Lim, Nicole Ngo-Niang-Ngo, Theresa Rossouw, Lisa M Frenkel, Barry R Lutz. “Hour-long assay for detection of drug resistance in HIV-1 cross subtypes using recombinase polymerase amplification integrated into paper-based oligonucleotide ligation” * Authors with equal contributions. The technologies developed for OLA for HIV are platforms that can be adapted to detect other diseases and conditions. Chapter 9 HOLA: OLA to detect a human allele (HLA-B*57:01) describes our adaptation of OLA-Simple technologies to detect human allele HLA-B*57:01, which confers Abacavir hypersensitivity. Chapter 10 Technologies in response to SARS-CoV-2 pandemic describes recent work on SARS-CoV-2 detection built upon the knowledge in Chapters 2 - 4 and 6 and my prior work on swab characterization and transfer methods during my first-year rotation. The SARS-CoV-2 lyophilized RT-PCR assay was evaluated at Duke University, the University of Zimbabwe, and a pediatric clinic at Georgetown University. Chapter 11 Fish-OLA – Species identification tool to regulate IUU describes the development of Fish-OLA, which is an adaptation of the OLA-Simple chemistry to detect the presence of the two biomarker SNPs unique to codfish from the Bering Sea and the Aleutian Islands. We conducted a blind study to evaluate the Fish-OLA on >200 specimens. Such tools could contribute to the management of illegal, unreported, and unregulated (IUU) fishing. Below are the publications, upcoming manuscripts, and patent applications based on the work in this area: PUBLICATIONS1) Jackson J Wallner, Ingrid A Beck, Nuttada Panpradist, Santiago Avila-Ríos, Humberto Valenzuela-Ponce, Maribel Soto-Nava, Barry R Lutz, Lisa M Frenkel. “Rapid, Near Point-of-Care Assay for HLA-B*57:01 Genotype Associated with Severe Hypersensitivity to Abacavir”. AIDS Research and Human Retroviruses. DOI: 10.1089/aid.2021.0103 2) Nuttada Panpradist, Qin Wang, Parker S Ruth, Jack H Kotnik, Amy K Oreskovic, Abraham Miller, Samuel WA Stewart, Justin Vrana, Peter D Han, Ingrid A Beck, Lea M Starita, Lisa M Frenkel, Barry R Lutz. “Simpler and faster Covid-19 testing: Strategies to streamline SARS-CoV-2 molecular assays.” EBiomedicine. 2021. PMID: 33582488. DOI: 10.1016/j.ebiom.2021.103236 3) Nuttada Panpradist, Robert Atkinson, Michael Roller, Enos Kline, Ian Hull, Qin Wang, Jack Henry Kotnik, Amy K Oreskovic, Crissa Bennett, Daniel Leon, Victoria Lyon, Peter D Han, Lea M Starita, Matthew Thompson, Barry R Lutz. “Harmony Covid-19: development and evaluation of ready-to-use reagents, hardware, and software package for point-of-care SARS-CoV-2 nucleic acid amplification test” Accepted to Science Advances 2021. DOI: 10.1101/2021.08.12.21261875. 4) Enos Kline, Nuttada Panpradist, Ian Hull, Qin Wang, Amy K Oreskovic, Peter D Han, Lea M Starita, Barry R Lutz. “Development of novel engineered polymerase and universal probe design to enable robust RT-LAMP for SARS-CoV-2 detection.” medRxiv. PMID: 34462755. DOI: 10.1101/2021.08.13.21261995. 5) Jason Hoffman, Matthew Hirano, Nuttada Panpradist, Joseph Breda, Parker Ruth, Yuanyi Xu, Jonathan Lester, Bichlien Nguyen, Luis Ceze, Shwetak N. Patel. “Passively Sensing SARS-CoV-2 RNA in Public Transit Buses. BioRXiv” https://doi.org/10.1101/2021.06.02.21258184 6) Andrew C. Hunt, James Brett Case, Young-Jun Park, Longxing Cao, Kejia Wu, Alexandra C. Walls, Zhuoming Liu, John E. Bowen, Hsien-Wei Yeh, Shally Saini, Louisa Helms, Yan Ting Zhao, Tien-Ying Hsiang, Tyler, N. Starr, Inna Goreshnik, Lisa Kozodoy, Lauren Carter, Rashmi Ravichandran, Lydia B. Green, Wadim L. Matochko, Christy A. Thomson, Bastain Vögeli, Antje Krüger-Gericke, Laura A. VanBlargan, Rita E. Chen, Baoling Ying, Adam L. Bailey, Natasha M. Kafai, Scott Boyken, Ajasja Ljubetič, Natasha Edman, George Ueda, Cameron Chow, Amin Addetia, Nuttada Panpradist, Michael Gale Jr, Benjamin S. Freedman, Barry R. Lutz, Jesse D. Bloom, Hannele Ruohola-Baker, Sean P. J. Whelan, Lance Stewart, Michael S. Diamond, David Veesler, Michael C. Jewett, David Baker. “Multivalent designed proteins protect against SARS-CoV-2 variants of concern” https://doi.org/10.1101/2021.07.07.451375 7) Nuttada Panpradist, Bhushan J Toley, Xiaohong Zhang, Samantha Byrnes, Joshua R Buser, Janet A Englund, Barry R Lutz. “Swab sample transfer for point-of-care diagnostics: characterization of swab types and manual agitation methods” 2014. PloS One. PMID: 25181250. DOI: 10.1371/journal.pone.0105786 MANUSCRIPT IN PREPARATION1) Nuttada Panpradist, Carolyn M Tarpey, Anita Wray, Parker S Ruth, Ingrid Spies, Barry R Lutz, Karl F Bohringer, Lorenz Hauser. “Fish-OLA: rapid and inexpensive population assignment for Pacific Cod (Gadus macrocephalus)” PATENT APPLICATIONS 1) Provisional Patent Application 63/165,029 filed 3/23/2021 Entitled: "SYSTEMS AND METHODS FOR DETECTING SARS-COV-2 RNA." 2) Provisional Patent Application 63/049,941 filed 7/9/2020 Entitled: "Kit for Sample Collection and Preparation for Amplification." 3) Provisional Patent Application 63/049,758 filed 7/9/2020 Entitled: "Amplification Device." Chapter 12 Summary of contributions and impact summarizes my contributions to the field and the impact of these works. In addition to the outcomes described in my thesis, I consulted other researchers on other projects, including diagnostics of cell-free DNA in urine samples and detection of TB drug metabolites. I was a visiting scholar at Stanford University and researched the dynamic host responses to pathogen infections via viral-inclusive single-cell RNASeq. Some of my contributions beyond thesis work are listed below. The complete list is available in Appendix 7 and Appendix 8. 1) Zhiyuan Yao, Fabio Zanini, Sathish Kumar, Marwah Karim, Sirle Saul, Nishank Bhalla, Nuttada Panpradist, Avery Muniz, Aarthi Narayanan, Stephen R Quake, Shirit Einav. “The transcriptional landscape of Venezuelan equine encephalitis virus infection” 2021. PLoS Neglected Diseases, DOI: 10.1371/journal.pntd.00093062) Amy Oreskovic, Nuttada Panpradist, Diana Marangu, M William Ngwane, Zanele P Magcaba, Sindiswa Ngcobo, Zinhle Ngcobo, David J Horne, Douglas PK Wilson, Adrienne E Shapiro, Paul K Drain, Barry R Lutz. “Diagnosing pulmonary tuberculosis using sequence-specific purification of urine cell-free DNA.”2021. JCM. PMID: 33789959; DOI: 10.1128/JCM.00074-21 3) Sylvia M LaCourse, Daniel Leon, Nuttada Panpradist, Barbra A Richardson, Elizabeth Maleche-Obimbo, Jerphason Mecha, Daniel Matemo, Jaclyn N Escudero, John Kinuthia, Barry Lutz, Grace John-Stewart. “Urine Biomarker Assessment of Infant Adherence to Isoniazid Prophylaxis” Pediatr Infect Dis J. 2021 Jan;40(1):e43-e45. DOI: 10.1097/INF.0000000000002936 4) Amy Oreskovic, Norman D Brault, Nuttada Panpradist, James J Lai, Barry R Lutz. “Analytical comparison of methods for extraction of short cell-free DNA from urine” The Journal of Molecular Diagnostics. 2019. PMID: 31442674. DOI: 10.1016/j.jmoldx.2019.07.002 Besides presenting conference proceedings, I have shared my expertise through other speaking platforms such as invited talks and lectures. Below are some highlights. The complete lists are available in Appendix 5 and Appendix 9.1) PHPT, Chiang Mai University, Thailand (2020) 2) Department of Medicine, University of Zimbabwe, Zimbabwe (2019) 3) KwaZulu-Natal Research Innovation and Sequencing Platform (KRISP), UKZN, Durban, South Africa (2018) 4) Michael Mayer Lab, University of Fribourg, Switzerland. (2017) 5) MEMS Research Unit, University of Mechanical Engineering, Chulalongkorn University, Bangkok, Thailand (2015) 6) PHPT, Chiang Mai University, Thailand (2015) 7) Department of Material Sciences, Silpakorn University, Thailand (2015

The transcriptional landscape of Venezuelan equine encephalitis virus (TC-83) infection.

Venezuelan Equine Encephalitis Virus (VEEV) is a major biothreat agent that naturally causes outbreaks in humans and horses particularly in tropical areas of the western hemisphere, for which no antiviral therapy is currently available. The host response to VEEV and the cellular factors this alphavirus hijacks to support its effective replication or evade cellular immune responses are largely uncharacterized. We have previously demonstrated tremendous cell-to-cell heterogeneity in viral RNA (vRNA) and cellular transcript levels during flaviviral infection using a novel virus-inclusive single-cell RNA-Seq approach. Here, we used this unbiased, genome-wide approach to simultaneously profile the host transcriptome and vRNA in thousands of single cells during infection of human astrocytes with the live-attenuated vaccine strain of VEEV (TC-83). Host transcription was profoundly suppressed, yet "superproducer cells" with extremely high vRNA abundance emerged during the first viral life cycle and demonstrated an altered transcriptome relative to both uninfected cells and cells with high vRNA abundance harvested at later time points. Additionally, cells with increased structural-to-nonstructural transcript ratio exhibited upregulation of intracellular membrane trafficking genes at later time points. Loss- and gain-of-function experiments confirmed pro- and antiviral activities in both vaccine and virulent VEEV infections among the products of transcripts that positively or negatively correlated with vRNA abundance, respectively. Lastly, comparison with single cell transcriptomic data from other viruses highlighted common and unique pathways perturbed by infection across evolutionary scales. This study provides a high-resolution characterization of the VEEV (TC-83)-host interplay, identifies candidate targets for antivirals, and establishes a comparative single-cell approach to study the evolution of virus-host interactions

Plate and paper formats for Capture, Denaturation and Detection (CDD) in the oligonucleotide ligation assay.

<p>(A) Plate CDD procedure. Products from the ligation step (both ligated and non-ligated products) are captured on a streptavidin-coated plate. Non-ligated probes are released during oligonucleotide denaturation, and ligated MUT and WT probes are then detected in sequential enzyme-based immunoassays (labeling by different detection antibodies, alkaline phosphatase yellow substrate development, optical density reading at 405nm, wash steps, tetramethylbenzidine (TMB) development, stop solution, and optical density reading at 450nm). (B) Paper CDD procedure. Similar to the plate CDD procedure, products from the ligation step (both ligated and non-ligated products) are captured. However, here the products are captured on paper strips by immobilized streptavidin. Non-ligated probes are released during oligonucleotide denaturation. Antibodies targeting the end-labels of the mutant (MUT) or wild-type (WT) probes have conjugated horseradish peroxidase (POD) that converts 3,3’ diazoaminobenzidine substrate (DAB) into brown precipitates. Signals were captured by the scanner (600 DPI). Reported signals represent capture spot intensity minus a background region from the strip.</p

Analysis of clinical specimens and plasmid standards by paper capture, denaturation, and detection (CDD) and plate CDD for mutations K103N and Y181C.

<p>Panels A and C show mutant (MUT) detection, and Panels B and D show wild-type (WT) detection. Sample optical density (OD) minus negative control OD (left y axis) for each specimen is shown in white/gray by rank along the x axis, from the lowest MUT OD, followed by the plasmid standards (0%, 5%, 50% MUT) performed in duplicate. Spot intensity minus background intensity (right y axis) for each specimen is shown in blue and green bars followed by the plasmid standards (0%, 5%, 50% MUT) performed in triplicate. Scanned images of the paper CDD detection strip are shown below each specimen’s signal data.</p

Analysis of clinical specimens and plasmid standards by paper capture, denaturation, and detection (CDD) and plate CDD for mutations M184V and G190A.

<p>Panels A and C show mutant (MUT) detection, and Panels B and D show wild-type (WT) detection. Sample optical density (OD) minus negative control OD (left y axis) for each specimen is shown in white/gray by rank along the x axis, from the lowest MUT OD, followed by the plasmid standards (0%, 5%, 50% MUT) performed in duplicate. Spot intensity minus background intensity (right y axis) for each specimen is shown in pink and orange bars followed by the plasmid standards (0%, 5%, 50% MUT) performed in triplicate. Scanned images of the paper CDD detection strip are shown below each specimen’s signal data.</p

Correlation plot of MUT Ratios determined by paper capture, denaturation, and detection (CDD) versus plate CDD.

<p>MUT and WT signals obtained by paper and plate CDD format OLA at codons K103N, Y181C, M184V and G190A from 26 clinical specimens were used to calculate MUT Ratios. The overall shape reflects the signal saturation of the paper CDD at high MUT concentration, as seen in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145962#pone.0145962.g003" target="_blank">3</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145962#pone.0145962.g005" target="_blank">5</a>. Results correlate strongly across clinical specimens and plasmid standards’ data (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145962#pone.0145962.s004" target="_blank">S4 Fig</a> for fitted correlation).</p

Comparison of paper Capture, Denaturation, and Detection (CDD) and plate CDD for analysis of a plasmid standard mixture series for mutation Y181C.

<p>(A) Scanned images of paper CDD MUT (left) and WT (right) detection (B) Mutant (MUT) detection (C) wild-type (WT) detection. Left axes: Specimen optical density (OD) minus negative control OD analyzed in duplicate by plate CDD (mean ± SE). Right axes: Specimen capture intensity minus background intensity analyzed in triplicate by paper CDD (mean ± SE). The ratio of mean signal intensities for 2% MUT and 0% MUT was 2.00 for plate CDD and 2.74 for paper CDD.</p

Comparison of manual agitation methods for swab transfer.

<p>(A) Schematic of action performed over a period of 1 second for different manual twirling methods. (B) Comparison of % organism recovery of PUR swabs using different twirling methods, which was calculated using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105786#pone.0105786.e016" target="_blank">Equation 5</a> in the text. (C) Schematic of the new forced flow syringe method. (D) Comparison of % organism recovery for PES and rayon swabs, using different twirling methods and the forced flow syringe method. * indicates statistically significant differences (Tukey-Kramer, α = 0.05).</p

Organism recovery for low-volume samples.

<p>(A) Schematic of the experimental set up. 15 µL <i>S. aureus</i>/TE (∼100, ∼10⁴, or ∼10⁶ CFU, equivalent to 500, 6×10⁴, or 4×10⁶<i>ldh1</i> copies, respectively, as measured by qPCR) was spiked onto the swab, which was then agitated in 128 µL lysis buffer using 10 second 1 Hz side twirl, and removed. (B) Comparison of the % Organism Recovery in four swabs at three different organism input numbers (mean ± SE, N = 5), which was calculated using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105786#pone.0105786.e016" target="_blank">Equation 5</a> in the text. (C) Comparison of the % Organism Recovery (mean ± SE; N = 5) using ∼10⁴ CFU/swab of <i>S. aureus</i> in the presence and absence of simulated nasal matrix (SNM).</p