A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of SARS-CoV-2 within nasopharyngeal and oropharyngeal swabs at Hampshire Hospitals NHS Foundation Trust

AbstractThe COVID-19 pandemic has illustrated the importance of rapid, accurate diagnostic testing for the effective triaging and cohorting of patients and timely tracking and tracing of cases. However, a surge in diagnostic testing quickly resulted in worldwide competition for the same sample preparation and real-time RT-PCR diagnostic reagents (rRT-PCR). Consequently, Hampshire Hospitals NHS Foundation Trust, UK sought to diversify their diagnostic portfolio by exploring alternative amplification chemistries including those that permit direct testing without RNA extraction. This study describes the validation of a SARS-CoV-2 RT-LAMP assay, which is an isothermal, autocycling, strand-displacement nucleic acid amplification technique which can be performed on extracted RNA, “RNA RT-LAMP” or directly from swab “Direct RT-LAMP”. Analytical specificity (ASp) of this new RT-LAMP assay was 100% and analytical sensitivity (ASe) was between 1×101 and 1×102 copies when using a synthetic DNA target. The overall diagnostic sensitivity (DSe) and specificity (DSp) of RNA RT-LAMP was 97% and 99% respectively, relative to the standard of care (SoC) rRT-PCR. When a CT cut-off of 33 was employed, above which increasingly, evidence suggests there is a very low risk of patients shedding infectious virus, the diagnostic sensitivity was 100%. The DSe and DSp of Direct-RT-LAMP was 67% and 97%, respectively. When setting CT cut-offs of ≤33 and ≤25, the DSe increased to 75% and 100%, respectively. Time from swab-to-result for a strong positive sample (CT < 25) was < 15 minutes. We propose that RNA RT-LAMP could replace rRT-PCR where there is a need for increase in throughput, whereas Direct RT-LAMP could be used as a screening tool for triaging patients into appropriate hospitals wards, at GP surgeries and in care homes, or for population screening to identify highly contagious individuals (“super shedders”). Direct RT-LAMP could also be used during times of high prevalence to save critical extraction and rRT-PCR reagents by “screening” out those strong positives from diagnostic pipelines.

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Real-time optical analysis of a colorimetric LAMP assay for SARS-CoV-2 in saliva with a handheld instrument improves accuracy compared to endpoint assessment

10.1101/2021.01.13.21249412 ◽

2021 ◽

Author(s):

Lena M. Diaz ◽

Brandon E. Johnson ◽

Daniel M. Jenkins

Keyword(s):

Nucleic Acid ◽

Quantitative Method ◽

Limit Of Detection ◽

Detection System ◽

Diagnostic Testing ◽

Rna Extraction ◽

Lamp Assay ◽

Nucleic Acid Amplification ◽

Clinical Samples ◽

Ph Indicator

AbstractControlling the course of the COVID-19 pandemic will require widespread deployment of consistent and accurate diagnostic testing of the novel coronavirus SARS-CoV-2. Ideally, tests should detect a minimum viral load, be minimally invasive, and provide a rapid and simple readout. Current FDA-approved RT-qPCR-based standard diagnostic approaches require invasive nasopharyngeal swabs and involve laboratory-based analyses that can delay results. Recently, a loop mediated isothermal nucleic acid amplification (LAMP) test that utilizes colorimetric readout received FDA approval. This approach utilizes a pH indicator dye to detect drop in pH from nucleotide hydrolysis during nucleic acid amplification. This method has only been approved for use with RNA extracted from clinical specimens collected via nasopharyngeal swabs. In this study, we developed a quantitative LAMP-based strategy to detect SARS-CoV-2 RNA in saliva. Our detection system distinguished positive from negative sample types using a handheld instrument that monitors optical changes throughout the LAMP reaction. We used this system in a streamlined LAMP testing protocol that could be completed in less than two hours to directly detect inactivated SARS-CoV-2 in minimally processed saliva that bypassed RNA extraction, with a limit of detection (LOD) of 50 genomes/reaction. The quantitative method correctly detected virus in 100% of contrived clinical samples spiked with inactivated SARS- CoV-2 at either 1X (50 genomes/reaction) or 2X (100 genomes/reaction) of the LOD. Importantly the quantitative method was based on dynamic optical changes during the reaction so was able to correctly classify samples that were misclassified by endpoint observation of color.

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Robust Saliva-Based RNA Extraction-Free One-Step Nucleic Acid Amplification Test for Mass SARS-CoV-2 Monitoring

Molecules ◽

10.3390/molecules26216617 ◽

2021 ◽

Vol 26 (21) ◽

pp. 6617

Author(s):

Eva Rajh ◽

Tina Šket ◽

Arne Praznik ◽

Petra Sušjan ◽

Alenka Šmid ◽

...

Keyword(s):

Oral Cavity ◽

Reverse Transcription ◽

Screening Program ◽

Quantitative Polymerase Chain Reaction ◽

Rna Extraction ◽

Diagnostic Sensitivity ◽

Nucleic Acid Amplification ◽

Nasopharyngeal Swab ◽

Viral Spread ◽

One Step

Early diagnosis with rapid detection of the virus plays a key role in preventing the spread of infection and in treating patients effectively. In order to address the need for a straightforward detection of SARS-CoV-2 infection and assessment of viral spread, we developed rapid, sensitive, extraction-free one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) tests for detecting SARS-CoV-2 in saliva. We analyzed over 700 matched pairs of saliva and nasopharyngeal swab (NSB) specimens from asymptomatic and symptomatic individuals. Saliva, as either an oral cavity swab or passive drool, was collected in an RNA stabilization buffer. The stabilized saliva specimens were heat-treated and directly analyzed without RNA extraction. The diagnostic sensitivity of saliva-based RT-qPCR was at least 95% in individuals with subclinical infection and outperformed RT-LAMP, which had at least 70% sensitivity when compared to NSBs analyzed with a clinical RT-qPCR test. The diagnostic sensitivity for passive drool saliva was higher than that of oral cavity swab specimens (95% and 87%, respectively). A rapid, sensitive one-step extraction-free RT-qPCR test for detecting SARS-CoV-2 in passive drool saliva is operationally simple and can be easily implemented using existing testing sites, thus allowing high-throughput, rapid, and repeated testing of large populations. Furthermore, saliva testing is adequate to detect individuals in an asymptomatic screening program and can help improve voluntary screening compliance for those individuals averse to various forms of nasal collections.

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Investigation of pooling strategies using clinical COVID-19 samples for more efficient diagnostic testing

10.1101/2020.08.10.20171819 ◽

2020 ◽

Author(s):

Samantha H Adikari ◽

Emily Z Alipio Lyon ◽

Attelia D Hollander ◽

Alina Deshpande ◽

Elizabeth Hong-Geller

Keyword(s):

Diagnostic Testing ◽

Rna Extraction ◽

Positive Sample ◽

Viral Rna ◽

Clinical Samples ◽

Extraction Column ◽

Diagnostic Assay ◽

Significant Difference ◽

Pooling Strategies ◽

Single Positive

When testing large numbers of clinical COVID-19 samples for diagnostic purposes, pooling samples together for processing can offer significant reductions in the materials, reagents, time, and labor needed. We have evaluated two different strategies for pooling independent nasopharyngeal swab samples prior to testing with an EUA-approved SARS-CoV-2 RT-qPCR diagnostic assay. First, in the Dilution Study, we assessed the assay's ability to detect a single positive clinical sample diluted in multiple negative samples before the viral RNA extraction stage. We observed that positive samples with Ct values at ~30 can be reliably detected in pools of up to 30 independent samples, and positive samples with Ct values at ~35 can be detected in pools of 5 samples. Second, in the Reloading Study, we assessed the efficacy of reloading QIAamp viral RNA extraction columns numerous times using a single positive sample and multiple negative samples. We determined that one RNA extraction column can be reloaded with up to 20 clinical samples (1 positive and 19 negatives) sequentially without any loss of signal in the diagnostic assay. Furthermore, we found there was no significant difference in assay readout whether the positive sample was loaded first or last in a series of 20 samples. These results demonstrate that different pooling strategies can lead to increased process efficiencies for COVID-19 clinical diagnostic testing.

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2359. Prospective Feasibility Study for Novel Ultrasensitive Multiplexed Immunoassay for Clostridiodes difficile Toxins A and B

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.2037 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S812-S813

Author(s):

Lauren Watson ◽

Michele L Zimbric ◽

Catherine Shaughnessy ◽

Shraddha Kale ◽

Jeff Debad ◽

...

Keyword(s):

Diagnostic Tests ◽

Standard Of Care ◽

Positive Sample ◽

Nucleic Acid Amplification ◽

Serum Samples ◽

Toxin A ◽

Toxin B ◽

Diagnostic Assays ◽

Stool Samples ◽

Multiplexed Immunoassay

Abstract Background The diagnosis of Clostridiodes difficile infection is challenging. A wide array of diagnostic tests are used in practice; however, each available test has important limitations. We examined the feasibility and analytical performance of a novel ultrasensitive multiplexed immunoassay designed by Meso Scale Diagnostics (MSD) compared with five current diagnostic assays for detection of C. difficile toxin A and B. Methods Stool, serum and urine samples from 44 admitted inpatients were collected within 72 hours of a standard of care nucleic acid amplification test (NAAT) result (23 positive, 21 negative). These specimens underwent five standard diagnostic assays: enzyme immunoassay for toxins A and B (EIA), cytotoxin cell assay, bacterial culture isolation, and two different NAATs to determine presence of viable C. difficile cells, toxins, and toxin-encoding genes (Table 1). The concentration (fg/mL) of toxin A and toxin B in all stool samples was then quantified using MSD’s multiplexed immunoassay (Table 1). Results At least one of the five standard diagnostic tests for C. difficile was positive in 16 of the 23 clinically positive patients. The MSD multiplex immunoassay detected toxin A and/or toxin B in 15 of these 16 samples and quantified low levels of toxin A in one clinically positive sample that was negative for all other tests. In contrast, only 2 of the 16 positive samples were positive by EIA, demonstrating the benefits of the ultrasensitive assay over standard immunoassay methods. All clinically negative specimens were negative in all tests. Toxin detection in urine and serum samples was negligible. In stool samples, the MSD test had an estimated sensitivity of 93% (95% CI: 70–99%) and specificity of 93% (95% CI: 78–98%) compared with the clinically used NAAT. Conclusion The MSD multiplex toxin assay is a feasible test to move forward for further evaluation. Ultimately, future studies should examine the performance of this test compared with standard of care in a prospective randomized trial assessing clinical outcomes. Disclosures All authors: No reported disclosures.

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2564. Clinical Validation of a Commercial LAMP Test for Ruling Out Malaria in Returning Travelers: A Prospective Diagnostic Trial

Open Forum Infectious Diseases ◽

10.1093/ofid/ofy209.172 ◽

2018 ◽

Vol 5 (suppl_1) ◽

pp. S72-S72 ◽

Cited By ~ 3

Author(s):

James Cheaveau ◽

Hong Nguyen ◽

Barbara Chow ◽

Hong Yuan Zhou ◽

Abu Naser Mohon ◽

...

Keyword(s):

Clinical Laboratory ◽

Cost Benefit Analysis ◽

Diagnostic Testing ◽

Cost Benefit ◽

Malaria Diagnosis ◽

Signs And Symptoms ◽

Nucleic Acid Amplification ◽

Analytical Sensitivity ◽

Benefit Analysis ◽

Diagnostic Trial

Abstract Background The mainstay of malaria diagnosis relies on rapid diagnostic tests (RDT) and Giemsa-stained microscopy both of which lack analytical sensitivity. This leads to repeat testing to rule out malaria. Nucleic acid amplification (NAT) methods are more sensitive, but testing requires technical proficiency beyond the average clinical laboratory. Methods We conducted a prospective diagnostic trial of the Meridian illumigene Malaria assay (LAMP) compared with reference microscopy and RDT (BinaxNOW Malaria) in returning travelers in Western Canada between June 2017 and January 2018. Returning travelers with signs and symptoms of fever were enrolled into the study. RDT, microscopy, and LAMP assays were performed simultaneously. To increase the yield of positive specimens for all species of malaria, retrospective specimens of Plasmodium vivax, P. ovale, and P. malariae species were supplemented. Real-time (RT)–PCR testing was performed on all specimens to resolve discrepancies. A cost–benefit analysis was performed. Results A total of 296 consecutive patients (50.7% male, mean age 32.5) were enrolled, most visiting friends and relatives (43.2%), traveling to Asia (48.4%), presenting with fever (88.9%), not taking prophylaxis (82.8%), and treated as outpatients (84.3%). In the prospective arm, LAMP had a sensitivity of 98.1% (95% CI 89.9–99.9) and a specificity of 97.6% (95% CI 95.2–99.0) versus microscopy. After discrepant resolution with RTPCR, LAMP had a sensitivity of 100% (95% CI 93.9–100) and a specificity of 100% (95% CI 98.7–100) versus microscopy. When including retrospective specimens, LAMP had a sensitivity of 98.7% (95% CI 92.7–99.9) and a specificity of 97.6% (95% CI 95.2–99.1) versus microscopy, and after discrepant resolution of this set, LAMP had a sensitivity of 100% (95% CI 95.5–100) and a specificity of 100% (95% CI 98.7–100). The rate of invalid tests with LAMP was 3.05%. After discrepant resolution, RDT had a sensitivity of 83.3% (95% CI 58.6–96.4) and a specificity of 96.2% (95% CI 93.2–98.1) versus microscopy. A cost–benefit analysis of reagents and labor suggests up to USD 13 savings per specimen using a revised algorithm with LAMP screening. Conclusion A novel, highly sensitive testing algorithm for malaria screening with associated cost savings in the nonendemic setting is proposed. Disclosures D. Pillai, Meridian Biosciences: None, Diagnostic testing material for study.

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Direct diagnostic testing of SARS-CoV-2 without the need for prior RNA extraction

10.1101/2020.05.28.20115220 ◽

2020 ◽

Cited By ~ 3

Author(s):

Shan Wei ◽

Esther Kohl ◽

Alexandre Djandji ◽

Stephanie Morgan ◽

Susan Whittier ◽

...

Keyword(s):

Limit Of Detection ◽

Point Of Care ◽

Low Cost ◽

Diagnostic Testing ◽

Rna Extraction ◽

Nucleic Acid Amplification ◽

Nasopharyngeal Swab ◽

Viral Transport ◽

Extraction Step ◽

Highly Sensitive

AbstractThe COVID-19 pandemic has resulted in an urgent global need for rapid, point-of-care diagnostic testing. Existing methods for nucleic acid amplification testing (NAAT) require an RNA extraction step prior to amplification of the viral RNA. This step necessitates the use of a centralized laboratory or complex and costly proprietary cartridges and equipment, and thereby prevents low-cost, scalable, point-of-care testing. We report the development of a highly sensitive and robust, easy-to-implement, SARS-CoV-2 test that utilizes isothermal amplification and can be run directly on viral transport media following a nasopharyngeal swab without the need for prior RNA extraction. Our assay provides visual results in 30 min with 85% sensitivity, 100% specificity, and a limit of detection (LoD) of 2.5 copies/μl, and can be run using a simple heat block.

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Rapid Multiplex Loop-Mediated Isothermal Amplification (m-LAMP) Assay for Differential Diagnosis of Leprosy and Post–Kala-Azar Dermal Leishmaniasis

American Journal of Tropical Medicine and Hygiene ◽

10.4269/ajtmh.19-0313 ◽

2021 ◽

Author(s):

Shweta Joshi ◽

Keerti K. Dixit ◽

Vanila Sharma ◽

V. Ramesh ◽

Ruchi Singh ◽

...

Keyword(s):

Differential Diagnosis ◽

Leishmania Donovani ◽

Clinical Manifestations ◽

Lamp Assay ◽

Isothermal Amplification ◽

Diagnostic Sensitivity ◽

Analytical Sensitivity ◽

Loop Mediated Isothermal Amplification ◽

Kala Azar ◽

Endemic Areas

Leprosy and post–kala-azar dermal leishmaniasis (PKDL) are co-endemic neglected tropical diseases often misdiagnosed because of close resemblance in their clinical manifestations. The test that aids in differential diagnosis of leprosy and PKDL would be useful in endemic areas. Here, we report development of a multiplex loop-mediated isothermal amplification (m-LAMP) assay for differential detection of Mycobacterium leprae and Leishmania donovani using a real-time fluorometer. The m-LAMP assay was rapid with a mean amplification time of 15 minutes, and analytical sensitivity of 1 fg for L. donovani and 100 fg for M. leprae. The distinct mean Tm values for M. leprae and L. donovani allowed differentiation of the two organisms in the m-LAMP assay. Diagnostic sensitivity of the assay was evaluated by using confirmed cases of leprosy (n = 40) and PKDL (n = 40) (tissue and slit aspirate samples). All the leprosy and PKDL samples used in this study were positive by organism-specific QPCR and loop-mediated isothermal amplification assays. The diagnostic sensitivity of the m-LAMP assay was 100% (95% CI: 91.2–100.0%) for detecting PKDL and 95% for leprosy (95% CI: 83.1–99.4%). Our m-LAMP assay was successfully used to detect both M. leprae and L. donovani in a patient coinfected with leprosy and macular PKDL. The m-LAMP assay is rapid, accurate, and applicable for differential diagnosis of leprosy versus PKDL, especially in endemic areas.

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Performance of Saliva, Oropharyngeal Swabs, and Nasal Swabs for SARS-CoV-2 Molecular Detection: A Systematic Review and Meta-analysis

10.1101/2020.11.12.20230748 ◽

2020 ◽

Cited By ~ 1

Author(s):

Rose A. Lee ◽

Joshua C. Herigon ◽

Andrea Benedetti ◽

Nira R. Pollock ◽

Claudia M. Denkinger

Keyword(s):

Meta Analysis ◽

Diagnostic Testing ◽

Rna Extraction ◽

High Sensitivity ◽

Nucleic Acid Amplification ◽

Sample Collection ◽

Specimen Collection ◽

Nasal Swabs ◽

The Impact ◽

Alternative Specimen

ABSTRACTBackgroundNasopharyngeal (NP) swabs are considered the highest-yield sample for diagnostic testing for respiratory viruses, including SARS-CoV-2. The need to increase capacity for SARS-CoV-2 testing in a variety of settings, combined with shortages of sample collection supplies, have motivated a search for alternative sample types with high sensitivity. We systematically reviewed the literature to understand the performance of alternative sample types compared to NP swabs.MethodsWe systematically searched PubMed, Google Scholar, medRxiv, and bioRxiv (last retrieval October 1st, 2020) for comparative studies of alternative specimen types [saliva, oropharyngeal (OP), and nasal (NS) swabs] versus NP swabs for SARS-CoV-2 diagnosis using nucleic acid amplification testing (NAAT). A logistic-normal random-effects meta-analysis was performed to calculate % positive alternative-specimen, % positive NP, and % dual positives overall and in sub-groups. The QUADAS 2 tool was used to assess bias.ResultsFrom 1,253 unique citations, we identified 25 saliva, 11 NS, 6 OP, and 4 OP/NS studies meeting inclusion criteria. Three specimen types captured lower % positives [NS (0.82, 95% CI: 0.73-0.90), OP (0.84, 95% CI: 0.57-1.0), saliva (0.88, 95% CI: 0.81 – 0.93)] than NP swabs, while combined OP/NS matched NP performance (0.97, 95% CI: 0.90-1.0). Absence of RNA extraction (saliva) and utilization of a more sensitive NAAT (NS) substantially decreased alternative-specimen yield.ConclusionsNP swabs remain the gold standard for diagnosis of SARS-CoV-2, although alternative specimens are promising. Much remains unknown about the impact of variations in specimen collection, processing protocols, and population (pediatric vs. adult, late vs. early in disease course) and head-to head studies of sampling strategies are urgently needed.

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Rapid Detection of Hepatitis B Virus in Blood Samples Using a Combination of Polymerase Spiral Reaction With Nanoparticles Lateral-Flow Biosensor

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2020.578892 ◽

2021 ◽

Vol 7 ◽

Author(s):

Lin Lin ◽

Jinshuai Guo ◽

Haiyang Liu ◽

Xiaofeng Jiang

Keyword(s):

Limit Of Detection ◽

Point Of Care ◽

Diagnostic Technique ◽

Lamp Assay ◽

Cross Reactivity ◽

Lateral Flow ◽

Nucleic Acid Amplification ◽

Analytical Sensitivity ◽

Blood Samples ◽

Nucleic Acid Amplification Technology

A rapid, highly sensitive, and robust diagnostic technique for point-of-care (PoC) testing can be developed using the combination of the nanoparticle-based lateral flow biosensors (LFB) and isothermal nucleic acid amplification technology. Here, we developed a polymerase spiral reaction (PSR) containing FITC-labeled DNA probes coupled with the nanoparticle-based LFB assay (PSR-LFB) to detect the amplified products to detect HBV visually. Under the optimized conditions, the PSR assay involved incubation of the reaction mixture for 20 min at 63°C, followed by visual detection of positive amplicons using LFB, which would generate a red test line based on the biotin/streptavidin interaction and immunoreactions, within 5 min. A cross-reactivity test revealed that the developed PSR-LFB assay showed good specificity for HBV and could distinguish HBV from other pathogenic microorganisms. For the analytical sensitivity, the limit of detection (LoD) of PSR-LFB assay was recorded as 5.4 copies/mL of HBV genomic DNA, which was ten-times more sensitive than qPCR and loop-mediated isothermal amplification (LAMP). Additionally, all the HBV-positive (29/82) samples, identified using ELISA, were also successfully detected by the PSR-LFB assay. We found that the true positive rate of the PSR-LFB assay was higher than that of qPCR (100 vs. 89.66%, respectively), as well as the LAMP assay (100 vs. 96.55%, respectively). Furthermore, the integrated procedure could be completed in 60 min, including the processing of the blood samples (30 min), an isothermal reaction (20 min), and result visualization (5 min). Thus, this PSR-LFB assay could be a potentially useful technique for PoC diagnosis of HBV in resource-limited countries.

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Multicenter Evaluation of the Cepheid Xpert Xpress SARS-CoV-2 Test

Journal of Clinical Microbiology ◽

10.1128/jcm.00926-20 ◽

2020 ◽

Vol 58 (8) ◽

Cited By ~ 32

Author(s):

Michael J. Loeffelholz ◽

David Alland ◽

Susan M. Butler-Wu ◽

Utsav Pandey ◽

Carlo Frederico Perno ◽

...

Keyword(s):

Respiratory Tract ◽

Lower Respiratory Tract ◽

Limit Of Detection ◽

Clinical Performance ◽

High Sensitivity ◽

Standard Of Care ◽

Nucleic Acid Amplification ◽

Therapeutic Interventions ◽

Nasopharyngeal Swab ◽

Analytical Sensitivity

ABSTRACT Nucleic acid amplification tests (NAATs) are the primary means of identifying acute infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Accurate and fast test results may permit more efficient use of protective and isolation resources and allow rapid therapeutic interventions. We evaluated the analytical and clinical performance characteristics of the Xpert Xpress SARS-CoV-2 (Xpert) test, a rapid, automated molecular test for SARS-CoV-2. Analytical sensitivity and specificity/interference were assessed with infectious SARS-CoV-2; other infectious coronavirus species, including SARS-CoV; and 85 nasopharyngeal swab specimens positive for other respiratory viruses, including endemic human coronaviruses (hCoVs). Clinical performance was assessed using 483 remnant upper- and lower-respiratory-tract specimens previously analyzed by standard-of-care (SOC) NAATs. The limit of detection of the Xpert test was 0.01 PFU/ml. Other hCoVs, including Middle East respiratory syndrome coronavirus, were not detected by the Xpert test. SARS-CoV, a closely related species in the subgenus Sarbecovirus, was detected by a broad-range target (E) but was distinguished from SARS-CoV-2 (SARS-CoV-2-specific N2 target). Compared to SOC NAATs, the positive agreement of the Xpert test was 219/220 (99.5%), and the negative agreement was 250/261 (95.8%). A third tie-breaker NAAT resolved all but three of the discordant results in favor the Xpert test. The Xpert test provided sensitive and accurate detection of SARS-CoV-2 in a variety of upper- and lower-respiratory-tract specimens. The high sensitivity and short time to results of approximately 45 min may impact patient management.

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