scholarly journals Rapid Isothermal Amplification for the Buccal Detection SARS-CoV-2 in the Context of Out-Patient COVID-19 Screening

2021 ◽  
Vol 10 (12) ◽  
pp. 2643
Author(s):  
Amar Bouam ◽  
Jean-Jacques Vincent ◽  
Elisabeth Le Glass ◽  
Lionel Almeras ◽  
Pierre-Yves Levy ◽  
...  
Keyword(s):  
Point Of Care ◽  
Sampling Procedure ◽  
Isothermal Amplification ◽  
The Self ◽  
Rt Pcr ◽  
Negative Results ◽  
Data Support ◽  
Buccal Swabs ◽  
Pcr Assays ◽  
Positive Results

A commercially available isothermal amplification of SARS-CoV-2 RNA was applied to self-collected saliva samples using dry dental cotton rolls, which were held in the mouth for two minutes. Of 212 tests, isothermal amplification yielded three (0.14%) invalid results, 120 (56.6%) positive results and 89 (42%) negative results. Compared to reference RT-PCR assays routinely performed simultaneously on nasopharyngeal swabs, excluding the three invalid isothermal amplification assays and one RT-PCR invalid assay, these figures indicated that 119/123 (96.7%) samples were positive in both methods and 85/85 samples were negative in both methods. Four positive buccal swabs which were missed by the isothermal amplification, exhibited Ct values of 26–34 in reference RT-PCR assays. Positive isothermal amplification detection was achieved in less than 10 min. Supervision of the self-sampling procedure was key to achieve these performances. These data support the proposal to use the protocol reported in this paper, including supervised buccal self-sampling, to screen people suspected of having COVID-19 at the point of care.

scholarly journals False-Negative Results in Point-of-Care Qualitative Human Chorionic Gonadotropin (hCG) Devices Due to Excess hCGβ Core Fragment

2009 ◽  
Vol 55 (7) ◽  
pp. 1389-1394 ◽  
Author(s):  
Ann M Gronowski ◽  
Mark Cervinski ◽  
Ulf-Håkan Stenman ◽  
Alison Woodworth ◽  
Lori Ashby ◽  
...  
Keyword(s):  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Point Of Care ◽  
False Negative ◽  
The Core ◽  
Core Fragment ◽  
Negative Results ◽  
False Negative Results ◽  
Positive Results ◽  
Urine Specimens

Abstract Background: During pregnancy, human chorionic gonadotropin (hCG) immunoreactivity in urine consists of intact hCG as well as a number of hCG variants including the core fragment of hCGβ (hCGβcf). We identified 3 urine specimens with apparent false-negative results using the OSOM® hCG Combo Test (Genzyme Diagnostics) qualitative hCG device and sought to determine whether an excess of 1 of the fragments or variants might be the cause of the interference. Methods: We measured concentrations of hCG variants in the urine from 3 patients with apparent false-negative hCG results. Purified hCG variants were added to urines positive for hCG and tested using the OSOM, ICON® 25 hCG (Beckman Coulter), and hCG Combo SP® Brand (Cardinal Health) devices. Results: Dilution of these 3 urine samples resulted in positive results on the OSOM device. Quantification of hCG variants in each of the 3 patient urine specimens demonstrated that hCGβcf occurred in molar excess of intact hCG. Addition of purified hCGβcf to hCG-positive urines caused false-negative hCG results using the OSOM and ICON qualitative urine hCG devices. Conclusions: Increased concentrations of hCGβcf can cause false-negative results on the OSOM and ICON qualitative urine hCG devices. .

scholarly journals Simultaneous Detection and Identification of Human Parainfluenza Viruses 1, 2, and 3 from Clinical Samples by Multiplex PCR

1998 ◽  
Vol 36 (5) ◽  
pp. 1388-1391 ◽  
Author(s):  
Juan E. Echevarría ◽  
Dean D. Erdman ◽  
Ella M. Swierkosz ◽  
Brian P. Holloway ◽  
Larry J. Anderson
Keyword(s):  
Simultaneous Detection ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Negative Results ◽  
Parainfluenza Viruses ◽  
Detection And Identification ◽  
Serotype 1 ◽  
Pcr Assays ◽  
Syncytial Virus ◽  
Human Parainfluenza Viruses

Reverse transcription (RT)-PCR assays have been widely described for use in the diagnosis of human parainfluenza viruses (HPIVs) and other respiratory virus pathogens. However, these assays are mostly monospecific, requiring separate amplifications for each HPIV type. In the present work, we describe multiplex RT-PCR assays that detect and differentiate HPIV serotypes 1, 2, and 3 in a combined reaction. Specifically, a mixture of three pairs of primers to conserved regions of the hemagglutinin-neuraminidase gene of each HPIV serotype was used for primary amplification, yielding amplicons with similar sizes. For typing, a second amplification was performed with a mixture of nested primers, yielding amplicons with sizes easily differentiated by agarose gel electrophoresis. A modified single-amplification RT-PCR assay with fluorescence-labeled nested primers, followed by analysis of the labeled products on an automated sequencing gel, was also evaluated. Fifteen temporally and geographically diverse HPIV isolates from the Centers for Disease Control and Prevention archives and 26 of 30 (87%) previously positive nasopharyngeal specimens (8 of 10 positive for HPIV serotype 1 [HPIV1], 9 of 10 positive for HPIV2, and 9 of 10 positive for HPIV3) were positive and were correctly typed by both assays. Negative results were obtained with naso- or oropharyngeal specimens and/or culture isolates of 33 unrelated respiratory tract pathogens, including HPIV4, enterovirus, rhinovirus, respiratory syncytial virus, adenovirus, influenza virus, and Streptococcus pneumoniae. Our multiplex RT-PCR assays provide sensitive, specific, and simplified tools for the rapid diagnosis of HPIV infections.

scholarly journals False-Negative Nasopharyngeal Swab RT-PCR Assays in Typical COVID-19: Role of Ultra-low-dose Chest CT and Bronchoscopy in Diagnosis

2020 ◽  
Author(s):  
Marco Marando ◽  
Adriana Tamburello ◽  
Pietro Gianella
Keyword(s):  
Low Dose ◽  
False Negative ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Oral Swab ◽  
Negative Results ◽  
False Negative Results ◽  
Polymerase Chain ◽  
Pcr Assays

On 11 March 2020, the WHO declared COVID-19 a pandemic and global health emergency. We describe the clinical features and role of ultra-low-dose chest computed tomography (CT) and bronchoscopy in the diagnosis of coronavirus disease (COVID-19). In our patient, who was highly suggestive clinically and radiologically for COVID-19, we had two false-negative results for nasopharyngeal and oral swab reverse-transcriptase polymerase chain reaction (RT-PCR) assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Eventually, we confirmed the diagnosis using bronchoscopy and bronchoalveolar lavage (BAL).

scholarly journals Development of a dual-gene loop-mediated isothermal amplification (LAMP) detection assay for SARS-CoV-2: A preliminary study

2020 ◽  
Author(s):  
Azeem Mehmood Butt ◽  
Shafiqa Siddique ◽  
Xiaoping An ◽  
Yigang Tong
Keyword(s):  
Point Of Care ◽  
Lamp Assay ◽  
Cross Reactivity ◽  
Isothermal Amplification ◽  
Nasopharyngeal Swab ◽  
Loop Mediated Isothermal Amplification ◽  
Qualitative Detection ◽  
Detection Assay ◽  
Testing Protocols ◽  
Positive Results

AbstractSevere acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) has emerged as a rapidly spreading global pathogen stressing the need for development of rapid testing protocols ever than before. The aim of present study was to develop a SARS-CoV-2 detection protocol which can be performed within minimal resources and timeframe. For this purpose, we implemented the reverse transcription loop-mediated isothermal amplification (RT-LAMP) methodology for the qualitative detection of SARS-CoV-2 RNA. In order to improve the detection capability, the RT-LAMP assay was developed to simultaneously amplify two viral genes: ORF1a and N. A total of 45 SARS-CoV-2 associated coronavirus disease 2019 (COVID-19) and 25 non-COVID-19 cases were enrolled. Viral RNA was extracted from the nasopharyngeal swab samples and analyzed simultaneously using PCR and RT-LAMP protocols. Overall, our SARS-CoV-2 dual gene RT-LAMP assay was found to be 95% accurate in detecting positive cases and showed no cross-reactivity or false-positive results in non-COVID-19 samples. Further evaluation on larger and multi-centric cohorts is currently underway to establish the diagnostic accuracy and subsequent implementation into clinical practice and at point-of-care settings.

scholarly journals Rapid Isothermal Amplification and Portable Detection System for SARS-CoV-2

2020 ◽  
Author(s):  
A. Ganguli ◽  
A. Mostafa ◽  
J. Berger ◽  
M. Aydin ◽  
F. Sun ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Detection System ◽  
Point Of Care ◽  
Rna Extraction ◽  
Clinical Diagnostics ◽  
Isothermal Amplification ◽  
Sample Collection ◽  
Rt Pcr ◽  
Point Of Use ◽  
Viral Transport Medium

AbstractThe COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay details and primer sequences become widely known, many laboratories could perform diagnostic tests using methods such as RT-PCR or isothermal RT-LAMP amplification. A key advantage of RT-LAMP based approaches compared to RT-PCR is that RT-LAMP is known to be robust in detecting targets from unprocessed samples. In addition, RT-LAMP assays are performed at a constant temperature enabling speed, simplicity, and point-of-use testing. Here, we provide the details of an RT-LAMP isothermal assay for the detection of SARS-CoV-2 virus with performance comparable to currently approved tests using RT-PCR. We characterize the assay by introducing swabs in virus spiked synthetic nasal fluids, moving the swab to viral transport medium (VTM), and using a volume of that VTM for performing the amplification without an RNA extraction kit. The assay has a Limit-of-Detection (LOD) of 50 RNA copies/μL in the VTM solution within 20 minutes, and LOD of 5000 RNA copies/μL in the nasal solution. Additionally, we show the utility of this assay for real-time point-of-use testing by demonstrating detection of SARS-CoV-2 virus in less than 40 minutes using an additively manufactured cartridge and a smartphone-based reader. Finally, we explore the speed and cost advantages by comparing the required resources and workflows with RT-PCR. This work could accelerate the development and availability of SARS-CoV-2 diagnostics by proving alternatives to conventional laboratory benchtop tests.Significance StatementAn important limitation of the current assays for the detection of SARS-CoV-2 stem from their reliance on time- and labor-intensive and laboratory-based protocols for viral isolation, lysis, and removal of inhibiting materials. While RT-PCR remains the gold standard for performing clinical diagnostics to amplify the RNA sequences, there is an urgent need for alternative portable platforms that can provide rapid and accurate diagnosis, potentially at the point-of-use. Here, we present the details of an isothermal amplification-based detection of SARS-CoV-2, including the demonstration of a smartphone-based point-of-care device that can be used at the point of sample collection.

scholarly journals A COVID-19 patient with multiple negative results for PCR assays outside Wuhan, China: a case report

2020 ◽  
Author(s):  
Li-Da Chen ◽  
Hao Li ◽  
Yu-Ming Ye ◽  
Zhi Wu ◽  
Ya-Ping Huang ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Clinical Manifestations ◽  
Vital Role ◽  
Human Interferon ◽  
Nasopharyngeal Swab ◽  
Lung Field ◽  
Rt Pcr ◽  
Negative Results ◽  
Lower Lung ◽  
Pcr Assays

Abstract Background: The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a public health emergency of major international concern. Real-time RT-PCR assays are recommended for diagnosis of COVID-19. Here we report a rare case of COVID-19 with multiple negative results for PCR assays outside Wuhan, China. Case presentation: A 32-year old male was admitted to our hospital because of 6 days of unexplained fever on January 29, 2020. He had come from Wuhan city 10 days before admission. 5 days before admission, no abnormality was noted in laboratory test, chest radiography, and nasopharyngeal swab test for the SARS-CoV-2 nucleic acid. The patient was treated with ibuprofen for alleviating fever. On admission, chest computed tomography showed multiple ground-glass opacities in right lower lung field. COVID-19 was suspected. 3 times of nasopharyngeal swab specimens were collected after admission. However, none of the specimens were positive. The patient was confirmed with COVID-19 after fifth SARS-CoV-2 nucleic acid test. He was treated with lopinavir/ritonavir, recombinant human interferon alfa-2b inhalation, methylprednisolone. After 18 days of treatment, he was discharged with improved symptoms, lung lesions and negative results of nasopharyngeal swab. Conclusion: This case reminds clinician that a patient with high clinical suspicion of COVID-19 but multiple negative RT-PCR result should not be taken out of isolation. A combination of patient’s exposure history, clinical manifestations, laboratory tests, and typical imaging findings plays a vital role in making preliminary diagnosis and guide early isolation and treatment. Repeat swab tests are helpful in diagnosis for this kind of patients.

scholarly journals Detecting SARS-CoV-2 at point of care: preliminary data comparing loop-mediated isothermal amplification (LAMP) to polymerase chain reaction (PCR)

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Marc F. Österdahl ◽  
Karla A. Lee ◽  
Mary Ni Lochlainn ◽  
Stuart Wilson ◽  
Sam Douthwaite ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Predictive Value ◽  
Point Of Care ◽  
Isothermal Amplification ◽  
Service Improvement ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Loop Mediated Isothermal Amplification ◽  
Polymerase Chain

Abstract Background A cost effective and efficient diagnostic tool for COVID-19 as near to the point of care (PoC) as possible would be a game changer in the current pandemic. We tested reverse transcription loop mediated isothermal amplification (RT-LAMP), a method which can produce results in under 30 min, alongside standard methods in a real-life clinical setting. Methods This prospective service improvement project piloted an RT-LAMP method on nasal and pharyngeal swabs on 21 residents of a high dependency care home, with two index COVID-19 cases, and compared it to multiplex tandem reverse transcription polymerase chain reaction (RT-PCR). We recorded vital signs of patients to correlate clinical and laboratory information and calculated the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of a single swab using RT-LAMP compared with the current standard, RT-PCR, as per Standards for Reporting Diagnostic Accuracy Studies (STARD) guidelines. Results The novel method accurately detected 8/10 RT-PCR positive cases and identified a further 3 positive cases. Eight further cases were negative using both methods. Using repeated RT-PCR as a “gold standard”, the sensitivity and specificity of a single novel test were 80 and 73% respectively. PPV was 73% and NPV was 83%. Incorporating retesting of low signal RT-LAMP positives improved the specificity to 100%. We also speculate that hypothermia may be a significant early clinical sign of COVID-19. Conclusions RT-LAMP testing for SARS-CoV-2 was found to be promising, fast and to work equivalently to RT-PCR methods. RT-LAMP has the potential to transform COVID-19 detection, bringing rapid and accurate testing to the PoC. RT-LAMP could be deployed in mobile community testing units, care homes and hospitals to detect disease early and prevent spread.

scholarly journals Comparative Diagnostic Performance of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Kit for the Rapid Detection of SARS-CoV-2

Pathogens ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1629
Author(s):  
Alexander Domnich ◽  
Andrea Orsi ◽  
Donatella Panatto ◽  
Vanessa De Pace ◽  
Valentina Ricucci ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Diagnostic Performance ◽  
Point Of Care ◽  
Lamp Assay ◽  
Laboratory Diagnosis ◽  
Isothermal Amplification ◽  
Molecular Diagnostic ◽  
Rt Pcr ◽  
Laboratory Equipment ◽  
Loop Mediated Isothermal Amplification

Although the reverse transcription-polymerase chain reaction (RT-PCR) is considered a standard-of-care assay for the laboratory diagnosis of SARS-CoV-2, several limitations of this method have been described. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is an alternative molecular assay and is potentially able to overcome some intrinsic shortcomings of RT-PCR. In this study, we evaluated the diagnostic performance of the novel HG COVID-19 RT-LAMP assay. In this retrospective analysis, a total of 400 routinely collected leftover nasopharyngeal samples with a known RT-PCR result were tested by means of the HG COVID-19 RT-LAMP assay. The overall sensitivity and specificity values of HG COVID-19 RT-LAMP versus RT-PCR were 97.0% (95% CI: 93.6–98.9%) and 98.5% (95% CI: 95.7–99.7%), respectively. Inter-assay agreement was almost perfect (κ = 0.96). Concordance was perfect in samples with high viral loads (cycle threshold < 30). The average time to a positive result on RT-LAMP was 17 min. HG COVID-19 RT-LAMP is a reliable molecular diagnostic kit for detecting SARS-CoV-2, and its performance is comparable to that of RT-PCR. Shorter turnaround times and the possibility of performing molecular diagnostics in the point-of-care setting make it a valuable option for facilities without sophisticated laboratory equipment.

scholarly journals Analysis of SARS-CoV-2 PCR and Chest CT Findings at a Single Facility in Tokyo (Machida Municipal Hospital)

2021 ◽  
Vol 2021 ◽  
pp. 1-5
Author(s):  
Taisuke Kazuyori ◽  
Akihito Sato ◽  
Yasuhiro Morimoto ◽  
Kazuyoshi Kuwano
Keyword(s):  
Chest Ct ◽  
Background Characteristics ◽  
Rt Pcr ◽  
Important Indicator ◽  
Negative Results ◽  
Ct Findings ◽  
Positive Rate ◽  
Polymerase Chain ◽  
The City ◽  
Positive Results

Introduction. The SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) test is useful for diagnosing COVID-19, and the RT-PCR positive rate is an important indicator for estimating the incidence rate and number of infections. COVID-19 pneumonia is also associated with characteristic findings on chest CT, which can aid in diagnosis. Methods. We retrospectively evaluated patient background characteristics, the number of cases, the positivity rate, and chest CT findings for positive and negative cases in 672 patients who underwent RT-PCR for suspected COVID-19 at our hospital between April 3 and August 28, 2020. In addition, we compared trends in the positive rates at approximately weekly intervals with trends in the number of new infections in Machida City, Tokyo. Results. The study included 323 men and 349 women, with a median age of 46 years (range: 1 month–100 years). RT-PCR findings were positive in 37 cases, and the positive rate was 5.51%. Trends in the positive rate at our hospital and the number of new COVID-19 cases in the city were similar during the study period. Among patients with positive results, 15 (40.5%) had chest CT findings, and 14 had bilateral homogeneous GGOs. Among patients with negative results, 190 had chest CT findings at the time of examination, and 150 were diagnosed with bacterial pneumonia or bronchitis, with main findings consisting of consolidations and centrilobular opacities. Only 11 of these patients exhibited bilateral homogeneous GGOs. Conclusion. Bilateral homogeneous GGOs are characteristic of COVID-19 pneumonia and may aid in the diagnosis of COVID-19.

scholarly journals Clinical and epidemiologic characteristics of inconclusive results in SARS-CoV-2 RT-PCR assays

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yong Kwan Lim ◽  
Oh Joo Kweon ◽  
Hye Ryoun Kim ◽  
Tae-Hyoung Kim ◽  
Mi-Kyung Lee
Keyword(s):  
Infection Prevention ◽  
Infection Prevention And Control ◽  
Rt Pcr ◽  
Subgenomic Rna ◽  
Pcr Assays ◽  
And Control ◽  
Positive Results ◽  
Epidemiologic Characteristics ◽  
Respiratory Specimens

Abstract Background Inconclusive results in SARS-CoV-2 molecular assays cause confusion among clinicians and delay appropriate infection prevention and control. In this study, we aimed to characterize the respiratory specimens associated with inconclusive SARS-CoV-2 molecular assay results. Methods We re-evaluated inconclusive specimens by 3 additional RT-PCR assays and attempted to detect subgenomic RNA (sgRNA) in these specimens. Results Among follow-up tests from confirmed SARS-CoV-2 cases, 36.3% of the inconclusive results were classified as presumptive positive results (45/124). However, none of the specimens from 36 screening cases was classified as a presumptive positive result. Among 160 inconclusive specimens, sgRNAs were detected in 78 samples (48.8%): 58 were confirmed cases (58/124, 46.8%) and 20 were screening cases (20/36, 55.6%). Conclusions The results of our study suggest the recommendation of considering inconclusive results as positive results for confirmed SARS-CoV-2 cases. In screening cases, viral remnants could be partially amplified in PCR assays, and these inconclusive results could be related to previous infections. In addition, sgRNAs were detected in about half of the inconclusive specimens; however, the clinical significance of sgRNA is not yet clear.

Sign in / Sign up

Export Citation Format

Share Document