scholarly journals Quantitative Isothermal Amplification on Paper Membranes using Amplification Nucleation Site Analysis

2022 ◽  
Author(s):  
Benjamin P. Sullivan ◽  
Yu-Shan Chou ◽  
Andrew T. Bender ◽  
Coleman D. Martin ◽  
Zoe G. Kaputa ◽  
...  
Keyword(s):  
Viral Load ◽  
Nucleic Acid ◽  
Infectious Diseases ◽  
Point Of Care ◽  
Nucleation Site ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Hiv Viral Load ◽  
Middle Income ◽  
Nucleation Sites

Quantitative nucleic acid amplification tests (qNAATs) are critical in treating infectious diseases, such as in HIV viral load monitoring or SARS-CoV-2 testing, in which viral load indicates viral suppression or infectivity. Quantitative PCR is the gold standard tool for qNAATs; however, there is a need to develop point-of-care (POC) qNAATs to manage infectious diseases in outpatient clinics, low- and middle-income countries, and the home. Isothermal amplification methods are an emerging tool for POC NAATs as an alternative to traditional PCR-based workflows. Previous works have focused on relating isothermal amplification bulk fluorescence signals to input copies of target nucleic acids for sample quantification with limited success. In this work, we show that recombinase polymerase amplification (RPA) reactions on paper membranes exhibit discrete fluorescent amplification nucleation sites. We demonstrate that the number of nucleation sites can be used to quantify HIV-1 DNA and RNA in less than 20 minutes. An image-analysis algorithm quantifies nucleation sites and determines the input nucleic acid copies in the range of 67-3,000 copies per reaction. We demonstrate a mobile phone-based system for image capture and onboard processing, illustrating that this method may be used at the point-of-care for qNAATs with minimal instrumentation.

Long-term dry storage of enzyme-based reagents for isothermal nucleic acid amplification in a porous matrix for use in point-of-care diagnostic devices

The Analyst ◽  
2020 ◽  
Vol 145 (21) ◽  
pp. 6875-6886 ◽  
Author(s):  
Sujatha Kumar ◽  
Ryan Gallagher ◽  
Josh Bishop ◽  
Enos Kline ◽  
Joshua Buser ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Glass Fiber ◽  
Point Of Care ◽  
Porous Matrix ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Isothermal Nucleic Acid Amplification ◽  
Dry Storage

Long-term dry storage of enzyme-based isothermal amplification reagents in glass fiber porous matrix for use in point-of-care devices.

scholarly journals A modular paper-and-plastic device for tuberculosis nucleic acid amplification testing in limited-resource settings

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Navjot Kaur ◽  
Joy S. Michael ◽  
Bhushan J. Toley
Keyword(s):  
Nucleic Acid ◽  
Point Of Care ◽  
Limited Resource ◽  
High Specificity ◽  
Nucleic Acid Amplification ◽  
Analytical Sensitivity ◽  
Preparation Technique ◽  
Middle Income ◽  
Clinical Sensitivity ◽  
The Cost

Abstract We present a prototype for conducting rapid, inexpensive and point-of-care-compatible nucleic acid amplification tests (NAATs) for tuberculosis (TB). The fluorescent isothermal paper-and-plastic NAAT (FLIPP-NAAT) uses paper-based loop mediated isothermal amplification (LAMP) for DNA detection. The cost of materials required to build a 12-test-zone device is $0.88 and the cost of reagents per reaction is $0.43. An inexpensive imaging platform enables filter-free fluorescence detection of amplified DNA using a cell-phone camera. FLIPP-NAAT can be operated by an untrained user and only requires a regular laboratory incubator as ancillary equipment. All reagents can be dry-stored in the device, facilitating storage and transportation without cold chains. The device design is modular and the assay demonstrated high specificity to Mycobacterium tuberculosis (Mtb), analytical sensitivity of the order of 10 copies of Mtb gDNA, and tolerance to complex samples. The clinical sensitivity and specificity of sputum-based FLIPP NAAT tests were 100% (zero false negatives) and 68.75% (five false positives), respectively (N = 30), using Xpert MTB/RIF assay as the reference standard. FLIPP-NAAT has the potential to provide affordable and accessible molecular diagnostics for TB in low- and middle-income countries, when used in conjunction with an appropriate sample preparation technique. Although demonstrated for the detection of TB, FLIPP-NAAT is a platform technology for amplification of any nucleic acid sequence.

scholarly journals Using a dual antibody point-of-care test with visual and digital reads to diagnose syphilis among people living with HIV in Botswana

2021 ◽  
pp. 095646242097563
Author(s):  
Irfaan Maan ◽  
David S Lawrence ◽  
Nametso Tlhako ◽  
Kehumile Ramontshonyana ◽  
Aamirah Mussa ◽  
...  
Keyword(s):  
Day Treatment ◽  
Point Of Care ◽  
Kappa Statistic ◽  
People Living With Hiv ◽  
Diagnostic Systems ◽  
Hiv Viral Load ◽  
Past Infection ◽  
Middle Income ◽  
Visual Reading ◽  
Active Syphilis

Syphilis data from low- and middle-income countries are lacking due to limited testing. Point-of-care tests (POCTs) have been promoted to expand testing but previously only included treponemal tests, which cannot distinguish active from past infection. We aimed to assess the feasibility of using a combined treponemal and non-treponemal POCT in HIV clinic patients in Gaborone, Botswana, and estimate syphilis prevalence in our clinic sample using this approach. We recruited 390 non-pregnant patients. Participants underwent a combined treponemal and non-treponemal POCT (Dual Path Platform (DPP®) Syphilis Screen and Confirm Assay (Chembio Diagnostic Systems)) on finger-prick blood sample and a questionnaire. Median age 45 years, 30% men, median CD4 count 565 cells/μL, and 91% had an HIV viral load <400 copies/mL. Five participants had active syphilis (1.3%, 95% CI 0.5–3.0%) and 64 had previous syphilis (16.4%, 95% CI 13.0–20.4%) using the DPP POCT. There was a reasonable level of agreement between digital and visual reading of the POCT (kappa statistic of 0.81); however, visual reading missed three active infections (60%). The level of active syphilis was similar to local antenatal data. The DPP POCT led to five participants with active syphilis being diagnosed and starting same-day treatment. The digital reader should be used.

scholarly journals Essential properties and pitfalls of colorimetric Reverse Transcription Loop-mediated Isothermal Amplification as a point-of-care test for SARS-CoV-2 diagnosis

2021 ◽  
Vol 27 (1) ◽  
Author(s):  
Bruna de Oliveira Coelho ◽  
Heloisa Bruna Soligo Sanchuki ◽  
Dalila Luciola Zanette ◽  
Jeanine Marie Nardin ◽  
Hugo Manuel Paz Morales ◽  
...  
Keyword(s):  
Viral Load ◽  
Internal Control ◽  
Reverse Transcription ◽  
Color Change ◽  
Point Of Care ◽  
Colorimetric Detection ◽  
False Negative ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification

Abstract Background SARS-CoV-2 Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) colorimetric detection is a sensitive and specific point-of-care molecular biology technique used to detect the virus in only 30 min. In this manuscript we have described a few nuances of the technique still not properly described in the literature: the presence of three colors clusters; the correlation of the viral load with the color change; and the importance of using an internal control to avoid false-negative results. Methods To achieve these findings, we performed colorimetric RT-LAMP assays of 466 SARS-CoV-2 RT-qPCR validated clinical samples, with color quantification measured at 434 nm and 560 nm. Results First we determinate a sensitivity of 93.8% and specificity of 90.4%. In addition to the pink (negative) and yellow (positive) produced colors, we report for the first time the presence of an orange color cluster that may lead to wrong diagnosis. We also demonstrated using RT-qPCR and RT-LAMP that low viral loads are related to Ct values > 30, resulting in orange colors. We also demonstrated that the diagnosis of COVID-19 by colorimetric RT-LAMP is efficient until the fifth symptoms day when the viral load is still relatively high. Conclusion This study reports properties and indications for colorimetric RT-LAMP as point-of-care for SARS-CoV-2 diagnostic, reducing false results, interpretations and optimizing molecular diagnostics tests application.

scholarly journals 2158. Cost-Effectiveness and Budget Impact of a Point-of-Care Nucleic Acid Amplification Test for Diagnosis of Group A Streptococcal Pharyngitis in the United States

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S732-S732
Author(s):  
James Karichu ◽  
Mindy Cheng ◽  
Joanna Sickler ◽  
Julie Munakata ◽  
S Pinar Bilir ◽  
...  
Keyword(s):  
United States ◽  
Cost Effectiveness ◽  
Nucleic Acid ◽  
Budget Impact ◽  
Point Of Care ◽  
Cost Savings ◽  
The United States ◽  
Nucleic Acid Amplification ◽  
Sensitivity Analyses ◽  
Group A

Abstract Background Group A streptococcal (GAS) pharyngitis is common in the United States (US). Each year, approximately 12 million people seek medical care for pharyngitis, accounting for ~2% of ambulatory care visits. The gold standard method for diagnosing GAS is culture. However, because culture is time intensive, rapid antigen detection tests (RADTs), with or without culture confirmation, are commonly used. Although RADTs provide results quickly, test sensitivity has been shown to be sub-optimal, which can lead to inappropriate treatment decisions. Recently, highly sensitive point-of-care nucleic acid amplification tests (POC NAAT), such as the cobas® Liat® System, have emerged. The objective of this study was to evaluate the cost-effectiveness (CE) and budget impact (BI) of adopting POC NAAT compared with RADT+culture confirmation to diagnose GAS pharyngitis from the US third-party payer perspective. Methods A decision-tree economic model was developed in Microsoft Excel to quantify costs and clinical outcomes associated with POC NAAT and RADT+culture over a one-year period. All model inputs were derived from published literature and public databases. Model outputs included costs and clinical effects measured as quality-adjusted life days (QALDs) lost. One-way and probabilistic sensitivity analyses were performed to assess the impact of uncertainty on results. Results CE analysis showed that POC NAAT would cost $44 per patient compared with $78 with RADT+ culture. POC NAAT was associated with fewer QALDs lost relative to RADT+ culture. Therefore, POC NAAT may be considered the “dominant” strategy (i.e., lower costs and higher effectiveness). Findings were robust in sensitivity analyses. BI analysis showed that adopting POC NAAT for diagnosis of GAS could yield cost-savings of 0.3% vs. current budget over 3 years. This is due to savings associated with testing, GAS-related complications, antibiotic treatment and treatment-associated complication costs. Conclusion Results suggest that adopting POC NAAT to diagnose GAS would be considered cost-effective and yield cost-savings for US payers relative to RADT+culture. Access to POC NAAT would be important to optimize appropriate GAS diagnosis and treatment decisions. Disclosures All authors: No reported disclosures.

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