Low saliva pH can yield false positives results in simple RT-LAMP-based SARS-CoV-2 diagnostic tests.
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ABSTRACT:
SUBMITTER: Uribe-Alvarez C
PROVIDER: S-EPMC8099103 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic demanded rapid diagnosis to isolate new COVID-19 cases and prevent disease transmission. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) rapidly became the gold standard for diagnosis. However, due to the high cost and delay of the results, other types of diagnosis were implemented, such as COVID-19 Ag Rapid Tests and Reverse Transcription Technique followed by Loop-Mediated isothermal Amplification (RT-LAMP). In this work, we validated the use of RT-LAMP in saliva samples rather than nasopharyngeal swabs, as the collection is more comfortable. First, we selected 5 primer sets based on the limit of detection for SARS-CoV-2 RNA, then validated their sensitivity and specificity in patient samples. A total of 117 samples were analyzed by fluorometric RT-LAMP and compared with qRT-PCR results. Our results show that the use of a high-sensitive primer ORF1-a, together with a low-sensitive primer set Gene E (time to threshold of 22.9 and 36.4 minutes, respectively, using 200 copies of viral RNA), achieved sensitivity in purified RNA from saliva samples of 95.2% (95% CI 76.1‒99.8) with 90.5% specificity (95% CI 69.6‒98.8) (n = 42).As RNA purification increases the turnaround time, we tested the outcome of RT-LAMP utilizing raw saliva samples without purification. The test achieved a sensitivity of 81.8% (95% CI 59.7‒94.8) and a specificity of 90.9% (95% CI 70.8‒98.8). As a result, the accuracy of 92.9% (95% CI 80.5‒98.5) in purified RNA-saliva samples was lowered to an acceptable level of 86.4% (95% CI 72.6‒94.8) in raw saliva. Although mass vaccination has been implemented, new strains and low vaccination progress helped to spread COVID-19. This study shows that it is feasible to track new COVID-19 cases in a large population with the use of raw saliva as sample in RT-LAMP assay which yields accurate results and offers a less invasive test.
| S-EPMC10391658 | biostudies-literature
Project description:Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a fast and convenient method to amplify and identify the transcripts of a targeted pathogen. We combined bioinformatic and experimental analyses to improve the RT-LAMP assay performance for COVID-19 diagnosis. First, we developed an improved algorithm to design LAMP primers targeting the nucleocapsid (N), membrane (M), and spike (S) genes of SARS-CoV-2. Next, we rigorously validated these new assays for their efficacy and specificity. Further, we demonstrated that multiplexed RT-LAMP assays could directly detect as low as a few copies of SARS-CoV-2 RNA in saliva, without the need of RNA isolation. Importantly, further testing using saliva samples from COVID-19 patients indicated that the new RT-LAMP assays were in total agreement in sensitivity and specificity with standard RT-qPCR. In summary, our new LAMP primer design algorithm along with the validated assays provide a fast and reliable method for the diagnosis of COVID-19 cases.
2021-04-22 | GSE173086 | GEO
Project description:Effective management of the COVID-19 pandemic requires widespread and frequent testing of the population for SARS-CoV-2 infection. Saliva has emerged as an attractive alternative to nasopharyngeal samples for surveillance testing as it does not require specialized personnel or materials for its collection and can be easily provided by the patient. We have developed a simple, fast, and sensitive saliva-based testing workflow that requires minimal sample treatment and equipment. After sample inactivation, RNA is quickly released and stabilized in an optimized buffer, followed by reverse transcription loop-mediated isothermal amplification (RT-LAMP) and detection of positive samples using a colorimetric and/or fluorescent readout. The workflow was optimized using 1,670 negative samples collected from 172 different individuals over the course of 6 months. Each sample was spiked with 50 copies/μL of inactivated SARS-CoV-2 virus to monitor the efficiency of viral detection. Using pre-defined clinical samples, the test was determined to be 100% specific and 97% sensitive, with a limit of detection of 39 copies/mL. The method was successfully implemented in a CLIA laboratory setting for workplace surveillance and reporting. From April 2021-February 2022, more than 30,000 self-collected samples from 755 individuals were tested and 85 employees tested positive mainly during December and January, consistent with high infection rates in Massachusetts and nationwide.
| S-EPMC9135294 | biostudies-literature
Project description:BackgroundCOVID-19 has become a major public health problem after the outbreak caused by SARS-CoV-2 virus. Great efforts to contain COVID-19 transmission have been applied worldwide. In this context, accurate and fast diagnosis is essential.MethodsIn this prospective study, we evaluated the clinical performance of three different RNA-based molecular tests - RT-qPCR (Charité protocol), RT-qPCR (CDC (USA) protocol) and RT-LAMP - and one rapid test for detecting anti-SARS-CoV-2 IgM and IgG antibodies.ResultsOur results demonstrate that RT-qPCR using the CDC (USA) protocol is the most accurate diagnostic test among those evaluated, while oro-nasopharyngeal swabs are the most appropriate biological sample. RT-LAMP was the RNA-based molecular test with lowest sensitivity while the serological test presented the lowest sensitivity among all evaluated tests, indicating that the latter test is not a good predictor of disease in the first days after symptoms onset. Additionally, we observed higher viral load in individuals who reported more than 3 symptoms at the baseline. Nevertheless, viral load had not impacted the probability of testing positive for SARS-CoV-2.ConclusionOur data indicates that RT-qPCR using the CDC (USA) protocol in oro-nasopharyngeal swabs samples should be the method of choice to diagnosis COVID-19.
| S-EPMC10170900 | biostudies-literature
Project description:BackgroundThe coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has killed millions of people worldwide. The current crisis has created an unprecedented demand for rapid test of SARS-CoV-2 infection.MethodsReverse transcription loop-mediated isothermal amplification (RT-LAMP) is a fast and convenient method to amplify and identify the transcripts of a targeted pathogen. However, the sensitivity and specificity of RT-LAMP were generally regarded as inferior when compared with the gold standard RT-qPCR. To address this issue, we combined bioinformatic and experimental analyses to improve the assay performance for COVID-19 diagnosis.FindingsFirst, by experimental screening as well as high-throughput sequencing studies, we discovered new primer features that impacted LAMP sensitivity and specificity. These features were then used to build an improved bioinformatics algorithm to design LAMP primers targeting SARS-CoV-2. We further rigorously validated these new assays for their efficacy and specificity. We demonstrated that multiplexed RT-LAMP assay could directly detect as low as 1.5 copies/µL of SARS-CoV-2 particles in saliva, without the need of RNA isolation. We further tested this ultra-sensitive and specific RT-LAMP assay using saliva samples from COVID-19 patients. Clinical validation results indicated that the new RT-LAMP assay was comparable to standard RT-qPCR in overall assay sensitivity and specificity.InterpretationIn summary, our new LAMP primer design algorithm along with the validated assays provide a fast and reliable method for the diagnosis of COVID-19 cases.FundingNational Institutes of Health.
| S-EPMC8674011 | biostudies-literature
Project description:The population genetic study of divergence is often carried out using a Bayesian genealogy sampler, like those implemented in ima2 and related programs, and these analyses frequently include a likelihood ratio test of the null hypothesis of no migration between populations. Cruickshank and Hahn (2014, Molecular Ecology, 23, 3133-3157) recently reported a high rate of false-positive test results with ima2 for data simulated with small numbers of loci under models with no migration and recent splitting times. We confirm these findings and discover that they are caused by a failure of the assumptions underlying likelihood ratio tests that arises when using marginal likelihoods for a subset of model parameters. We also show that for small data sets, with little divergence between samples from two populations, an excellent fit can often be found by a model with a low migration rate and recent splitting time and a model with a high migration rate and a deep splitting time.
| S-EPMC4615538 | biostudies-literature
Project description:Background Non-invasive prenatal testing (NIPT) has had an incomparable triumph in prenatal diagnostics in the last decade. Over 1400 research articles have been published, predominantly praising the advantages of this test. Methods The present study identified among the 1400 papers 24 original and one review paper, which were suited to re-evaluate the efficacy of > 750,000 published NIPT-results. Special attention was given to false-positive and false-negative result-rates. Those were discussed under different aspects—mainly from a patient-perspective. Results A 27: 1 rate of false-positive compared to false-negative NIPT results was found. Besides, according to all reported, real-positive, chromosomally aberrant NIPT cases, 90% of those would have been aborted spontaneously before birth. These findings are here discussed under aspects like (i) How efficient is NIPT compared to first trimester screening? (ii) What are the differences in expectations towards NIPT from specialists and the public? and (iii) There should also be children born suffering from not by NIPT tested chromosomal aberrations; why are those never reported in all available NIPT studies? Conclusions Even though much research has been published on NIPT, unbiased figures concerning NIPT and first trimester screening efficacy are yet not available. While false positive rates of different NIPT tests maybe halfway accurate, reported false-negative rates are most likely too low. The latter is as NIPT-cases with negative results for tested conditions are yet not in detail followed up for cases with other genetic or teratogenic caused disorders. This promotes an image in public, that NIPT is suited to replace all invasive tests, and also to solve the problem of inborn errors in humans, if not now then in near future. Overall, it is worth discussing the usefulness of NIPT in practical clinical application. Particularly, asking for unbiased figures concerning the efficacy of first trimester-screening compared to NIPT, and for really comprehensive data on false-positive and false-negative NIPT results. Supplementary Information The online version contains supplementary material available at 10.1186/s13039-022-00612-2.
| S-EPMC9392255 | biostudies-literature
Project description:Point-of-care diagnostic technologies are becoming more widely available for production species. Here, we describe the application of reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect the matrix (M) gene of influenza A virus in swine (IAV-S). M-specific LAMP primers were designed based on M gene sequences from IAV-S isolated in the USA between 2017 and 2020. The LAMP assay was incubated at 65 °C for 30 min, with the fluorescent signal read every 20 s. The assay's limit of detection (LOD) was 20 M gene copies for direct LAMP of the matrix gene standard, and 100 M gene copies when using spiked extraction kits. The LOD was 1000 M genes when using cell culture samples. Detection in clinical samples showed a sensitivity of 94.3% and a specificity of 94.9%. These results show that the influenza M gene RT-LAMP assay can detect the presence of IAV in research laboratory conditions. With the appropriate fluorescent reader and heat block, the assay could be quickly validated as a low-cost, rapid, IAV-S screening tool for use on farms or in clinical diagnostic labs.
| S-EPMC10051247 | biostudies-literature
Project description:Lymphedema is the chronic enlargement of tissue due to inadequate lymphatic function. Diagnosis is made by history and physical examination and confirmed with lymphoscintigraphy. The purpose of this study was to assess the accuracy of lymphoscintigraphy for the diagnosis of lymphedema and to determine characteristics of patients with false-negative tests.Individuals referred to our lymphedema program with "lymphedema" between 2009 and 2016 were analyzed. Subjects were assessed by history, physical examination, and lymphoscintigraphy. Patient age at presentation, duration of lymphedema, location of disease, gender, previous infections, and lymphedema type were analyzed.The study included 227 patients (454 limbs); lymphedema was diagnosed clinically in 169 subjects and confirmed by lymphoscintigraphy in 162 (117 primary, 45 secondary; 96% sensitivity). Fifty-eight patients were thought to have a condition other than lymphedema, and all had negative lymphoscintigrams (100% specificity). A subgroup analysis of the 7 individuals with lymphedema clinically, but normal lymphoscintigrams, showed that all had primary lymphedema; duration of disease and infection history were not different between true-positive and false-negative lymphoscintigram results (P = 0.5). Two patients with a false-negative test underwent repeat lymphoscintigraphy, which then showed lymphatic dysfunction consistent with lymphedema.Lymphoscintigraphy is very sensitive and specific for lymphedema. All patients with false-negative studies had primary lymphedema. A patient with a high clinical suspicion of lymphedema and a normal lymphoscintigram should be treated conservatively for the disease and undergo repeat lymphoscintigraphy.
| S-EPMC5548565 | biostudies-other
Project description:Rising SARS-CoV-2 cases, testing delays, and the risk of pre-symptomatic and asymptomatic transmission provided the impetus for an in-house rapid testing program. Employees and their household contacts were encouraged to self-collect saliva samples that were pooled for routine testing using an established colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay. In brief, individual or a maximum of four saliva samples were pooled and heat-inactivated to render microorganisms, especially SARS-CoV-2, non-infectious prior to being added to RT-LAMP assay tubes containing either the human sample control gene, RNase P, or a region of the SARS-CoV-2 gene, ORF1ab. During the second wave of SARS-CoV-2 infections in November 2020, two samples from an employee and a member of their household tested positive via RT-LAMP within two days of each other. A delayed clinical qRT-PCR test confirmation of both individuals 5 days later underscored the power of routine rapid testing with within-the-hour turnaround times. Workplace rapid testing programs using RT-LAMP are flexible in their design, have a reduced cost compared to qRT-PCR, may involve non-invasive self-saliva collection for increased safety for the testing personnel, and can be performed with minimal training.
| S-EPMC8871197 | biostudies-literature