Molecular point-of-care tests: efficiency of RT-LAMP in the rapid diagnosis of COVID-19 to assess hospital access.

Background: Safe hospital access needs rapid testing for SARS-CoV-2 to enable rationale use of limited resources. The current standard method for Coronavirus detection is the RT-qPCR. This study aimed to determine the diagnostic performance of the new rapid RT-LAMP test, compared to RT-qPCR, and his efficiency for rapid hospital access through the Emergency Department (E.D.). Method(s): 1576 UTM nasopharyngeal swabs, collected in E.D., have been tested for SARS-CoV-2 infection, using a kit RTLAMP. The same samples were also analyzed with a traditional RT-qPCR assay and the results have been compared in terms of sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). Result(s): The assay has demonstrated a sensitivity of 73.3% (95%CI: 62.4/82.0) and specificity of 87.1% (95%CI: 85.3/88.7), PPV 22.1%, NPV 98.5%. Conclusion(s): ICGENE RNA RT-LAMP kit (ICGENEHEALTH;Enbiotech, Angri, Salerno, Italy) efficiently exclude the presence of infection and reliably detects infectious patients (with Ct<30). RNA RT-LAMP could replace rRTPCR where there is the need to rapidly identify potentially contagious individuals, but its low PPV suggests that positive results should be confirmed by a reference method.Copyright © 2022 EDIZIONI MINERVA MEDICA.

A real-time monitoring platform of colorimetric LAMP for developing rapid visual detection kits of SARS-CoV-2

Visual detection of nucleic acids is important to diagnose the serious acute infectious diseases such as coronavirus disease 2019 (COVID-19). During this pandemic, reliable visual detection kits have been in high demand for screening and prevention of the virus. While developing these visual detection kits, a real-time monitoring platform is usually applied to study the amplification and detection processes of nucleic acids and optimize the detecting conditions. Herein, we developed a real-time monitoring platform of colorimetric loop-mediated isothermal amplification (LAMP) to investigate the amplification and detection processes of nucleic acids. Using this platform, we could obtain the real-time amplification curves, and optimize the reaction temperature, color change, and detection time. Based on the optimized conditions, a visual detection kit for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was successfully developed with a sensitivity of 102 copies µL−1 in 12 min. This real-time monitoring platform has advantages of simple construction, steady performance, high sensitivity, and outstanding anti-pollution capability, and could replace the traditional colorimetric methods by photographing and reading values. This platform would accelerate the development of visual detection kits for colorimetric LAMP, help to explore the amplification and transcription of nucleic acids, and provide support for the prevention of emerging biological threats. © 2023

Evaluation of RT-LAMP Assay for Rapid Detection of SARS-CoV-2.

OBJECTIVE: To evaluate the accuracy of the reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in community or primary-care settings. METHOD: We systematically searched the Web of Science, Embase, PubMed, and Cochrane Library databases. We conducted quality evaluation using ReviewManager software (version 5.0). We then used MetaDisc software (version 1.4) and Stata software (version 12.0) to build forest plots, along with a Deeks funnel plot and a bivariate boxplot for analysis. RESULT: Overall, the sensitivity, specificity, and diagnostic odds ratio were 0.79, 0.97, and 328.18, respectively. The sensitivity for the subgroup with RNA extraction appeared to be higher, at 0.88 (0.86-0.90), compared to the subgroup without RNA extraction, at 0.50 (0.45-0.55), with no significant difference in specificity. CONCLUSION: RT-LAMP assay exhibited high specificity regarding current SARS-CoV-2 infection. However, its overall sensitivity was relatively moderate. Extracting RNA was found to be beneficial in improving sensitivity.

The Utilization of the LAMP Method in SARS-CoV2 Detection as an Alternative Diagnostic

SARS-CoV2 requires full attention from all countries because this disease can cause death in patients with comorbid diseases. Though some countries already put the endemic status for COVID19, the emerging cases due to new variants should be prevented by mass tracing. Extraction test RT-qPCR is still the gold standard in declaring someone infected with COVID-19. However, the high cost of financing in RT-qPCR is the biggest problem in the diagnostic test process. Loop-mediated Isothermal Amplification (LAMP) is an inexpensive, accurate, and fast method that amplifies the nucleic acid by using four or six different primers. By doing some modification and enhancement, LAMP method assay and its modified technique could replace RT-qPCR as a rapid, sensitive, low-cost assay when there is a need for increased sample throughput by semi-quantitative visual detection. The LAMP method could promote faster and more economic tracing methods in the dense population. © 2022 UPM Press. All rights reserved.

A diagnostic accuracy study comparing RNA LAMP, direct LAMP, and rapid antigen testing from nasopharyngeal swabs.

Introduction: During the coronavirus disease 2019 (COVID-19) pandemic, the early detection and isolation of individuals infected with severe acute respiratory syndrome coronavirus disease 2 (SARS-CoV-2) through mass testing can effectively prevent disease transmission. SARS-CoV-2 nucleic acid rapid detection based on loop-mediated isothermal amplification (LAMP) may be appropriate to include in testing procedures. Methods: We used 860 nasopharyngeal specimens from healthcare workers of Huashan Hospital and COVID-19 patients collected from April 7th to 21st, 2022, to assess the clinical diagnostic performance of the LAMP assay marketed by Shanghai GeneSc Biotech and compared it to the result of a rapid antigen test (RAT) head-to-head. Results: Overall, the diagnostic performance of LAMP assay and RAT were as follows. The LAMP assay represented higher sensitivity and specificity than RAT, especially in the extracted RNA samples. The sensitivity was 70.92% and 92.91% for direct LAMP and RNA-LAMP assay, respectively, while the specificity was 99.86% and 98.33%. The LAMP assay had overall better diagnostic performance on the specimens with relatively lower C t values or collected in the early phase (≤7 days) of COVID-19. The combination of LAMP assay and RAT improved diagnostic efficiency, providing new strategies for rapidly detecting SARS-CoV-2. Conclusion: The LAMP assay are suitable for mass screenings of SARS-CoV-2 infections in the general population.

An isothermal lab-on-phone test for easy molecular diagnosis of SARS-CoV-2 near patients and in less than 1 hour.

OBJECTIVES: The performance of a new point-of-care CE-IVD-marked isothermal lab-on-phone COVID-19 assay was assessed in comparison to a gold standard real-time reverse transcriptase-PCR method. METHODS: The study was conducted following a nonprobability sampling of ≥16-year-old volunteers from three different laboratories, using direct mouthwash (N = 24) or nasopharyngeal (N = 191) clinical samples. RESULTS: The assay demonstrated 95.19% sensitivity and 100% specificity for detection of SARS-CoV-2 in direct nasopharyngeal crude samples and 78.95% sensitivity and 100% specificity in direct mouthwash crude samples. It also successfully detected currently predominant SARS-CoV-2 variants of concern (Beta B.1.351, Delta B.1.617.2, and Omicron B.1.1.529) and demonstrated to be inert against potential cross-reactions of other common respiratory pathogens that cause infections that present similar symptoms to COVID-19. CONCLUSION: This lab-on-phone pocket-sized assay relies on an isothermal amplification of SARS-CoV-2's N and E genes, taking just 50 minutes from sample to result, with only 2 minutes of hands-on time. It presents good performance when using direct nasopharyngeal crude samples, enabling a low-cost, real-time, rapid, and accurate identification of SARS-CoV-2 infections at the point of care, which is important for both clinical management and population screening, as a tool to break the chain of transmission of COVID-19 pandemic, especially in low-resources environments.

SIMPLE RNA EXTRACTION METHOD FOR REVERSE TRANSCRIPTION LOOP-MEDIATED AMPLIFICATION DETECTION OF SARS-COV-2

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a robust and cost-effective assay for rapid diagnosis of SARS-CoV-2 compared to reverse transcription quantitative (RT-q) PCR. The study evaluated the performance of RT-LAMP technique that incorporated a simple Chelex 100 resin-based RNA extraction step for SARS-CoV-2 detection targeting virus E (encoding envelope protein) and RdRP (encoding RNA-dependent RNA polymerase). Using primer sets for E and RdRP, the developed RT-LAMP assay had a limit of detection (LOD) of 1 copy/µl transcribed RNA. For nasopharyngeal and oropharyngeal swab samples (n = 58), in comparison to the gold standard RT-qPCR (amplifying E and RdRP) sensitivity, specificity, positive predictive value, and negative predictive value of SARS-CoV-2 RT-LAMP assay targeting E gene was 88% (95% confidence interval (CI): 75-96%), 87% (95% CI: 59-98%), 99% (95% CI: 97-100%), and 28% (95% CI: 14-48%), respectively and for RdRP gene was 67% (95% CI: 51-98%), 87% (95% CI: 59-98%), 100% (95% CI: 96-100%), and 12% (95% CI: 8-18%), respectively. The whole process of RT-LAMP assay was completed within ~60 minutes. This developed RT-LAMP method for on-site COVID-19 detection should be useful in resource limited settings. © 2022, SEAMEO TROPMED Network. All rights reserved.

Development of a colorimetric RT-LAMP assay for the detection of SARS-COV-2 isolated from Oman.

INTRODUCTION: A rapid and sensitive COVID-19 diagnostic test is required to aid in the prevention and control of the current COVID-19 pandemic spread. We developed a colorimetric, rapid, and sensitive RT-LAMP assay for the diagnosis of COVID-19 viral infection. METHODOLOGY: Complete genome sequences of 41 SARS-CoV-2 isolates from Oman were used in this study. Three primer sets (CoV_S1, CoV_S2, CoV_M1) were developed from all Omani SARS-CoV-2 genome sequences available at the time, targeting the spike protein gene and the M gene. The primer set (CoV_S1) was found to be the most sensitive and specific among the three designed sets. The sensitivity and specificity of the assay were compared to that of qRT-PCR. Direct testing of SARS-CoV-2 spiked saliva with the developed assay was evaluated. Lyophilized colorimetric assays were stored at room temperature and 4 °C and their ability to detect positive samples were tested for a period of 8 weeks. RESULTS: The RT-LAMP assay was validated by testing 145 COVID-19 clinical samples with a sensitivity of 96.9% and specificity of 94.7% when compared to the validated qRT-PCR assay. The assay specificity was tested against SARS-CoV Frankfurt 1 RNA virus and avian coronaviruses as they tested negative with the developed assay. The assay was lyophilized and managed to detect the positive samples colorimetrically when stored at 4 °C for up to 8 weeks. CONCLUSIONS: The assay can be utilized in its current form as a screening assay with the advantages of being simpler, quicker, and cheaper than the qRT-PCR.

Comparative Evaluation of Cartridge-Based Abbott ID NOW Test With Probe-Based Real-Time Reverse Transcription Polymerase Chain Reaction Assay for Detection of SARS-CoV-2.

BACKGROUND: The gold standard test for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recommended by WHO is real-time reverse transcription polymerase chain reaction (RT-PCR), which has a turnaround time of five to six hours. Abbott ID NOW (Abbott Diagnostics Scarborough, Inc., Scarborough, ME, USA), the cartridge-based loop-mediated isothermal amplification (LAMP) assay, was approved by FDA for Emergency Use Authorization as rapid point of care testing. The present study was planned to evaluate the performance of the cartridge-based Abbott ID NOW test by comparing it to the currently used standard probe-based real-time RT-PCR method for detection of SARS-CoV-2. METHODOLOGY: A cross-sectional study was conducted in a tertiary care hospital in the eastern part of India after getting institutional ethics committee (IEC) approval. Two hundred fifty-nine cases of various age groups of both sexes who were advised for testing for SARS-CoV-2 were included in the study. Nasopharyngeal swabs were collected according to protocol advisory by the Indian Council of Medical Research (ICMR), India. Dry swabs were sent for Abbott ID NOW testing and swabs in viral transport medium were sent for probe-based RT-PCR assay using the CoviPath kit (Thermo Fisher Scientific, Bangalore, India). The data were collected and statistical analysis was performed using Statistical Package for Social Sciences (SPSS) (IBM Corp., Armonk, NY, USA). Sensitivity, specificity, positive and negative predictive values for ID NOW were calculated taking RT-PCR as the gold standard.  Results: Out of 259 patients enrolled in the study, 49% were symptomatic for coronavirus disease 2019 (COVID-19). The prevalence rate of SARS-CoV-2 was 20.84% among the study population. Sensitivity and specificity, positive and negative predictive values of ID NOW test in comparison to RT-PCR assay was found to be 87%, 98%, 92.1% and 96.8% respectively. ID NOW detected seven out of 54 (12.9%) cases as false negative who were found to be positive with RT-PCR, with mean Ct value of the target genes >34. CONCLUSIONS: In this study the overall sensitivity for ID NOW assay was found to be lower, but specificity, positive and negative predictive values were found to be higher. It had the highest correlation to RT-PCR among symptomatic patients and at higher viral loads. Due to the ease of use and shortest result time for detecting COVID-19, ID NOW test could be used as a point-of-care test. But for all tests, the results should be interpreted according to the clinical and epidemiological context.

Role of Laboratory Medicine in SARS-CoV-2 Diagnostics. Lessons Learned from a Pandemic.

Since the 2019 novel coronavirus outbreak began in Wuhan, China, diagnostic methods in the field of molecular biology have been developing faster than ever under the vigilant eye of world's research community. Unfortunately, the medical community was not prepared for testing such large volumes or ranges of biological materials, whether blood samples for antibody immunological testing, or salivary/swab samples for real-time PCR. For this reason, many medical diagnostic laboratories have made the switch to working in the field of molecular biology, and research undertaken to speed up the flow of samples through laboratory. The aim of this narrative review is to evaluate the current literature on laboratory techniques for the diagnosis of SARS-CoV-2 infection available on pubmed.gov, Google Scholar, and according to the writers' knowledge and experience of the laboratory medicine. It assesses the available information in the field of molecular biology by comparing real-time PCR, LAMP technique, RNA sequencing, and immunological diagnostics, and examines the newest techniques along with their limitations for use in SARS-CoV-2 diagnostics.

Emergent Carotid Artery Stenting Following Intravenous Alteplase Infusion After Rapid Negative Diagnosis for COVID-19 by Loop-Mediated Isothermal Amplification Assay.

BACKGROUND: During the coronavirus disease 2019 (COVID-19) pandemic, a rapid screening method for COVID-19 detection is needed to decide the appropriate strategy to treat stroke patients. In acute ischemic stroke treatment, the efficacy and safety of emergent carotid artery stenting (eCAS) for hyperacute ischemic stroke (hAIS) due to internal carotid artery stenosis (ICS) have not been sufficiently established. CASE DESCRIPTION: A 71-year-old man with hAIS caused by severe ICS was treated via intravenous alteplase infusion. The patient underwent screening for COVID-19 by the loop-mediated isothermal amplification (LAMP) assay shortly after arrival at our institution. The LAMP result was obtained within 90 minutes, during intravenous alteplase infusion, and turned out to be negative. The symptom of hemiplegia worsened during alteplase infusion, and he, therefore, underwent eCAS after administration of aspirin (200 mg). Recanalization was achieved successfully by eCAS, and dual antiplatelet therapy and argatroban were administrated following eCAS. Hemorrhagic complications or restenosis/occlusion of the carotid artery were not observed. He was discharged without neurologic deficits 15 days following eCAS. Because of the rapid negative diagnosis for COVID-19 using the LAMP method, eCAS could be performed following standard procedures, along with infectious defense, without delay. CONCLUSIONS: This case report suggests that eCAS for hAIS due to ICS following intravenous alteplase can be an effective treatment, along with appropriate antiplatelet medication and management in select patients. During the COVID-19 pandemic, the LAMP assay for COVID-19 detection might be a suitable diagnostic strategy preceding stroke treatment because of the rapid turnaround time.

Road toward rapid-molecular point of care test to detect novel SARS-coronavirus 2019 (COVID-19): Review from updated literature.

Coronavirus disease 2019 (COVID-19) named by the WHO as a result of the global public health emergency. COVID-19 is caused by a new coronavirus named as novel coronavirus (2019-nCOV). From the first case reported in December 2019 it is now a pandemic situation and a major public health emergency. The COVID-19 transmission rate is very high, infecting two to three persons on average with contact to an already infected person. There is a need for the health system, specially in developing countries such as in Pakistan, to combat such a novel disease by rapid, accurate, and high quality diagnostic testing in order to screen suspected cases and also surveillance of the disease. A rapid, accurate and low-cost diagnostic point-of-care device is needed for timely diagnosis of COVID-19 and is essential to combat such outbreaks for compelling preventive measures against the disease spread. This review is to highlight the importance of point-of-care diagnostics device for robust and accurate diagnosis of COVID-19 in physician offices and other urgent healthcare-type settings and encourage academics and stake holders towards advancement in order to control outbreaks and develop the public health surveillance system.

2019 Novel Coronavirus Disease (COVID-19): Paving the Road for Rapid Detection and Point-of-Care Diagnostics.

We believe a point-of-care (PoC) device for the rapid detection of the 2019 novel Coronavirus (SARS-CoV-2) is crucial and urgently needed. With this perspective, we give suggestions regarding a potential candidate for the rapid detection of the coronavirus disease 2019 (COVID-19), as well as factors for the preparedness and response to the outbreak of the COVID-19.