scholarly journals Sensitivity evaluation of 2019 novel coronavirus (SARS-CoV-2) RT-PCR detection kits and strategy to reduce false negative

2020 ◽  
Author(s):  
Yunying Zhou ◽  
Fengyan Pei ◽  
Li Wang ◽  
Huailong Zhao ◽  
Huanjie Li ◽  
...  
Keyword(s):  
Viral Load ◽  
Nucleic Acid ◽  
Prevention And Control ◽  
False Negative ◽  
Infection Prevention And Control ◽  
Nucleic Acid Detection ◽  
Rt Pcr ◽  
Current Standard ◽  
Asymptomatic Patients ◽  
And Control

ABSTRACTAn ongoing outbreak of pneumonia associated with SARS-CoV-2 has now been confirmed globally. In absence of effective vaccines, infection prevention and control through diagnostic testing and quarantine is critical. Early detection and differential diagnosis of respiratory infections increases the chances for successful control of COVID-19 disease. The nucleic acid RT-PCR test is regarded as the current standard for molecular diagnosis with high sensitivity. However, the highest specificity confirmation target ORF1ab gene is considered to be less sensitive than other targets in clinical application. In addition, a large amount of recent evidence indicates that the initial missed diagnosis of asymptomatic patients with SARS-CoV-2 and discharged patients with “re-examination positive” may be due to low viral load, and the ability of rapid mutation of coronavirus also increases the rate of false negative results. We aimed to evaluate the sensitivity of different nucleic acid detection kits so as to make recommendations for the selection of validation kit, and amplify the suspicious result to be reportable positive by means of simple continuous amplification, which is of great significance for the prevention and control of the current epidemic and the discharge criteria of low viral load patients.

scholarly journals Sensitivity evaluation of 2019 novel coronavirus (SARS-CoV-2) RT-PCR detection kits and strategy to reduce false negative

2020 ◽  
Author(s):  
Yunying Zhou ◽  
Fengyan Pei ◽  
Li Wang ◽  
Huailong Zhao ◽  
Huanjie Li ◽  
...  
Keyword(s):  
Viral Load ◽  
Nucleic Acid ◽  
Prevention And Control ◽  
False Negative ◽  
Infection Prevention And Control ◽  
Nucleic Acid Detection ◽  
Rt Pcr ◽  
Mixed Sample ◽  
And Control ◽  
Selection Of

Abstract Background: In absence of effective vaccines, infection prevention and control of SARS-CoV-2 through diagnostic testing and quarantine is critical. Early detection and differential diagnosis of respiratory infections increases the chances for successful control of COVID-19 disease. The nucleic acid RT-PCR test is regarded as the current standard for molecular diagnosis with high sensitivity. However, the highest specificity confirmation target ORF1ab gene is considered to be less sensitive than other targets in clinical application. In addition, a large amount of recent evidence indicates that the initial missed diagnosis of asymptomatic patients with SARS-CoV-2 and discharged patients with “re-examination positive” may be due to low viral load, and the ability of rapid mutation of SARS-CoV-2 also increases the rate of false negative results. Moreover, the current used mixed sample nucleic acid detection is helpful to seek out the early community transmission of SARS-CoV-2, but the detection kit needs ultra-high detection sensitivity. Methods: From January to March 2020, 10 confirmed specimens of 2019-nCoV were recruited from three 2019-nCOV nucleic acid testing center. Five different amplification kits with three different primers and probes sources were selected. RT-PCR and continuous amplification of nasopharyngeal and oropharyngeal swabs and environmental specimens were performed to validate the sensitivity of the commercially available Nucleic acid test kits. Results: The results showed that ORF1ab gene can still be reported as positive at 1:10 dilution and the N gene even at 1:40 dilution with kit-1 through the verification of multiple positive samples, While other kits have less sensitive. The results in the suspicious range of weakly positive nasopharyngeal and oropharyngeal swabs and environmental specimens could be reported as positive after another re-amplification.Conclusions: Through evaluate the sensitivity of different nucleic acid detection kits, this study provide direct evidence for the selection of kits for mixed sample detection or make recommendations for the selection of validation kit, which is of great significance for the prevention and control of the current epidemic and the discharge criteria of low viral load patients.

scholarly journals Sensitivity evaluation of 2019 novel coronavirus (SARS-CoV-2) RT-PCR detection kits and strategy to reduce false negative

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0241469
Author(s):  
Yunying Zhou ◽  
Fengyan Pei ◽  
Mingyu Ji ◽  
Li Wang ◽  
Huailong Zhao ◽  
...  
Keyword(s):  
Viral Load ◽  
Nucleic Acid ◽  
False Negative ◽  
Detection Sensitivity ◽  
Nucleic Acid Detection ◽  
Rt Pcr ◽  
Current Standard ◽  
Mixed Sample ◽  
Asymptomatic Patients ◽  
Selection Of

The early detection and differential diagnosis of respiratory infections increase the chances for successful control of COVID-19 disease. The nucleic acid RT-PCR test is regarded as the current standard for molecular diagnosis. However, the maximal specificity confirmation target ORF1ab gene is considered to be less sensitive than other targets in clinical application. In addition, recent evidence indicated that the initial missed diagnosis of asymptomatic patients with SARS-CoV-2 and discharged patients with “re-examination positive” might be due to low viral load, and the ability of rapid mutation of SARS-CoV-2 also increases the rate of false-negative results. Moreover, the mixed sample nucleic acid detection is helpful in seeking out the early community transmission of SARS-CoV-2 rapidly, but the detection kit needs ultra-high detection sensitivity. Herein, the lowest detection concentration of different nucleic acid detection kits was evaluated and compared to provide direct evidence for the selection of kits for mixed sample detection or make recommendations for the selection of validation kit, which is of great significance for the prevention and control of the current epidemic and the discharge criteria of low viral load patients.

scholarly journals August 2020 Interim EuGMS guidance to prepare European Long-Term Care Facilities for COVID-19

2020 ◽  
Vol 11 (6) ◽  
pp. 899-913 ◽  
Author(s):  
Hubert Blain ◽  
Yves Rolland ◽  
Jos M. G. A. Schols ◽  
Antonio Cherubini ◽  
Stéphanie Miot ◽  
...  
Keyword(s):  
Prevention And Control ◽  
Infection Prevention ◽  
Infection Prevention And Control ◽  
Rt Pcr ◽  
Negative Effects ◽  
Term Care ◽  
Staff Members ◽  
Care Facilities ◽  
And Control

Abstract Purpose The European Geriatric Medicine Society (EuGMS) is launching a second interim guidance whose aim is to prevent the entrance and spread of COVID-19 into long-term care facilities (LTCFs). Methods The EuGMS gathered experts to propose a guide of measures to prevent COVID-19 outbreaks in LTCFs. It is based on the specific features of SARS-CoV-2 transmission in LTCFs, residents’ needs, and on experiences conducted in the field. Results Asymptomatic COVID-19 residents and staff members contribute substantially to the dissemination of COVID-19 infection in LTCFs. An infection prevention and control focal point should be set up in every LTCF for (1) supervising infection prevention and control measures aimed at keeping COVID-19 out of LTCFs, (2) RT-PCR testing of residents, staff members, and visitors with COVID-19 symptoms, even atypical, and (3) isolating subjects either infected or in contact with infected subjects. When a first LCTF resident or staff member is infected, a facility-wide RT-PCR test–retest strategy should be implemented for detecting all SARS-CoV-2 carriers. Testing should continue until no new COVID-19 cases are identified. The isolation of residents should be limited as much as possible and associated with measures aiming at limiting its negative effects on their mental and somatic health status. Conclusions An early recognition of symptoms compatible with COVID-19 may help to diagnose COVID-19 residents and staff more promptly. Subsequently, an earlier testing for SARS-CoV-2 symptomatic and asymptomatic LTCF staff and residents will enable the implementation of appropriate infection prevention and control. The negative effects of social isolation in residents should be limited as much as possible.

scholarly journals A case of SARS-CoV-2 carrier for 32 days with several times false negative nucleic acid tests

2020 ◽  
Author(s):  
Lingjie Song ◽  
Guibao Xiao ◽  
Xianqin Zhang ◽  
Zhan Gao ◽  
Shixia Sun ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Clinical Symptoms ◽  
Early Stage ◽  
Antiviral Treatment ◽  
Immune Therapy ◽  
False Negative ◽  
Clinical Manifestations ◽  
Nucleic Acid Detection ◽  
Rt Pcr ◽  
Nucleic Acid Tests

AbstractIn 2019, a novel coronavirus (SARS-CoV-2) was first discovered in Wuhan, Hubei, China, causing severe respiratory disease in humans, and has been identified as a public health emergency of international concern. With the spread of the virus, there are more and more false negative cases of RT-PCR nucleic acid detection in the early stage of potential infection. In this paper, we collected the epidemiological history, clinical manifestations, outcomes, laboratory results and images of a SARS-CoV-2 carrier with no significant past medical history. The patient was quarantined because of her colleague had been diagnosed. After the onset of clinical symptoms, chest CT results showed patchy ground-glass opacity (GGO) in her lungs, but it took a total of nine nucleic acid tests to confirm the diagnosis, among which the first eight RT-PCR results were negative or single-target positive. In addition to coughing up phlegm during her stay in the hospital, she did not develop chills, fever, abdominal pain, diarrhea and other clinical symptoms. Since initial antiviral treatment, the lung lesions were absorbed. But the sputum nucleic acid test was still positive. In combination with antiviral and immune therapy, the patient tested negative for the virus. Notably, SARS-CoV-2 was detected only in the lower respiratory tract samples (sputum) throughout the diagnosis and treatment period. This is a confirmed case of SARS-CoV-2 infection with common symptoms, and her diagnosis has undergone multiple false negatives, suggesting that it is difficult to identify certain carriers of the virus and that such patients may also increase the spread of the SARS-CoV-2.

scholarly journals Rational use of SARS-CoV-2 polymerase chain reaction tests within institutions caring for the vulnerable

F1000Research ◽  
2020 ◽  
Vol 9 ◽  
pp. 671
Author(s):  
Tom A. Yates ◽  
Graham S. Cooke ◽  
Peter MacPherson
Keyword(s):  
Polymerase Chain Reaction ◽  
Prevention And Control ◽  
Infection Prevention ◽  
False Negative ◽  
Careful Consideration ◽  
Infection Prevention And Control ◽  
Chain Reaction ◽  
Test Characteristics ◽  
Polymerase Chain ◽  
And Control

Institutions such as hospitals and nursing or long-stay residential homes accommodate individuals at considerable risk of mortality should they acquire SARS-CoV-2 infection. In these settings, polymerase chain reaction tests play a central role in infection prevention and control. Here, we argue that both false negative and false positive tests are possible and that careful consideration of the prior probability of infection and of test characteristics are needed to prevent harm. We outline evidence suggesting that regular systematic testing of asymptomatic and pre-symptomatic individuals could play an important role in reducing transmission of SARS-CoV-2 within institutions. We discuss how such a programme might be organised, arguing that frequent testing and rapid reporting of results are particularly important. We highlight studies demonstrating that polymerase chain reaction testing of pooled samples can be undertaken with acceptable loss of sensitivity, and advocate such an approach where test capacity is limited. We provide an approach to calculating the most efficient pool size. Given the current limitations of tests for SARS-CoV-2 infection, physical distancing and meticulous infection prevention and control will remain essential in institutions caring for vulnerable people.

scholarly journals Estimation of conjunctival swab and nasopharyngeal swab specimens for viral nucleic acid detection in Coronavirus disease 2019 patients to compare the viral load

2021 ◽  
Vol 4 ◽  
pp. 2
Author(s):  
Jaya Kaushik ◽  
Vikas Marwah ◽  
Ankita Singh ◽  
Y. V. K. Chaitanya ◽  
Rajeev Mohan Gupta ◽  
...  
Keyword(s):  
Viral Load ◽  
Nucleic Acid ◽  
Statistical Correlation ◽  
Nucleic Acid Detection ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Viral Nucleic Acid ◽  
Ocular Manifestations ◽  
Conjunctival Swab ◽  
Polymerase Chain

Objectives: The purpose of the study was to detect the presence of viral ribonucleic acid of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in conjunctival swab along with nasopharyngeal swab specimens of Coronavirus disease 2019 (COVID-19) patients. Material and Methods: Thirty COVID-19 patients with at least one sample positive for real-time reverse transcription-polymerase chain reaction for SARS-CoV-2 in nasopharyngeal swab with the presence or absence of ocular manifestations were included in the study. The conjunctival swab along with nasopharyngeal swab of each patient was collected and sent to microbiology lab for evaluation and analysis of viral nucleic acid to assess the viral load. Results: Out of 30 patients, 21 patients (70%) were males and the remaining nine patients (30%) were females. Mean age of the patients in the study was 44.80 ± 15.37 years. One patient had conjunctivitis as ocular manifestation. Two (6.7%) out of 30 patients were positive for RT-PCR SARS-CoV-2 in the conjunctival swab. There was no statistical correlation between nasopharyngeal swab and conjunctival swab positivity using Pearson’s correlation coefficient (r) = 0.010; P = 0.995 (>0.05). Conclusion: The results of the study revealed that SARS-CoV-2 can also be detected in conjunctival swabs of confirmed cases of COVID-19 patients. Although, in comparison to nasopharyngeal and throat swabs the rate of detection of SARS-CoV-2 in conjunctival swabs is relatively less, still diligent care and precautions should be practiced during the ophthalmic evaluation of COVID-19 patients.

scholarly journals Assessment of the Diagnostic Ability of Four Detection Methods Using Three Sample Types of COVID-19 Patients

2021 ◽  
Vol 11 ◽  
Author(s):  
Fei Yu ◽  
Guoliang Xie ◽  
Shufa Zheng ◽  
Dongsheng Han ◽  
Jiaqi Bao ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcription ◽  
Detection Methods ◽  
Nucleic Acid Detection ◽  
Sample Type ◽  
Rt Pcr ◽  
Chain Reactions ◽  
Polymerase Chain Reactions ◽  
Oropharyngeal Swab ◽  
And Control

BackgroundViral nucleic acid detection is considered the gold standard for the diagnosis of coronavirus disease 2019 (COVID-19), which is caused by SARS-CoV-2 infection. However, unsuitable sample types and laboratory detection kits/methods lead to misdiagnosis, which delays the prevention and control of the pandemic.MethodsWe compared four nucleic acid detection methods [two kinds of reverse transcription polymerase chain reactions (RT-PCR A: ORF1ab and N testing; RT-PCRB: only ORF1ab testing), reverse transcription recombinase aided amplification (RT-RAA) and droplet digital RT-PCR (dd-RT-PCR)] using 404 samples of 72 hospitalized COVID-19 patients, including oropharyngeal swab (OPS), nasopharyngeal swabs (NPS) and saliva after deep cough, to evaluate the best sample type and method for SARS-CoV-2 detection.ResultsAmong the four methods, dd-RT-PCR exhibited the highest positivity rate (93.0%), followed by RT-PCR B (91.2%) and RT-RAA (91.2%), while the positivity rate of RT-PCR A was only 71.9%. The viral load in OPS [24.90 copies/test (IQR 15.58-129.85)] was significantly lower than that in saliva [292.30 copies/test (IQR 20.20-8628.55)] and NPS [274.40 copies/test (IQR 33.10-2836.45)]. In addition, if OPS samples were tested alone by RT-PCR A, only 21.4% of the COVID-19 patients would be considered positive. The accuracy of all methods reached nearly 100% when saliva and NPS samples from the same patient were tested simultaneously.ConclusionsSARS-CoV-2 nucleic acid detection methods should be fully evaluated before use. High-positivity rate methods such as RT-RAA and dd-RT-PCR should be considered when possible. Furthermore, saliva after deep cough and NPS can greatly improve the accuracy of the diagnosis, and testing OPS alone is not recommended.

scholarly journals ddPCR: a more sensitive and accurate tool for SARS-CoV-2 detection in low viral load specimens

2020 ◽  
Author(s):  
Tao Suo ◽  
Xinjin Liu ◽  
Jiangpeng Feng ◽  
Ming Guo ◽  
Wenjia Hu ◽  
...  
Keyword(s):  
Viral Load ◽  
Limit Of Detection ◽  
False Negative ◽  
Digital Pcr ◽  
Clinical Samples ◽  
Double Blind Study ◽  
Rt Pcr ◽  
Current Standard ◽  
Double Blind ◽  
Sensitivity Specificity

AbstractReal time fluorescent quantitative PCR (RT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. However, due to the low viral load in patient throats and the limitations of RT-PCR, significant numbers of false negative reports are inevitable, which results in failure to timely diagnose, early treat, cut off transmission, and assess discharge criteria. To improve this situation, an optimized droplet digital PCR (ddPCR) was used for detection of SARS-CoV-2, which showed that the limit of detection of ddPCR is significantly lower than that of RT-PCR. We further explored the feasibility of ddPCR to detect SARS-CoV-2 nucleic acid from 77 clinical throat swab samples, including 63 suspected outpatients with fever and 14 supposed convalescents who were about to discharge after treatment, and compared with RT-PCR in terms of the diagnostic accuracy. In this double-blind study, we tested, surveyed subsequently and statistically analyzed 77 clinical samples. According to our study, 26 samples from COVID-19 patients with RT-PCR negative were detected as positive by ddPCR. No FPRs of RT-PCR and ddPCR were observed. The sensitivity, specificity, PPV, NPV, NLR and accuracy were improved from 40% (95% CI: 27–55%), 100% (95% CI: 54–100%), 100%, 16% (95% CI: 13–19%), 0.6 (95% CI: 0.48–0.75) and 47% (95% CI: 33–60%) for RT-PCR to 94% (95% CI: 83–99%), 100% (95% CI: 48–100%), 100%, 63% (95% CI: 36–83%), 0.06 (95% CI: 0.02–0.18) and 95% (95% CI: 84–99%) for ddPCR, respectively. Moreover, 14 (42.9 %) convalescents still carry detectable SARS-CoV-2 after discharge. Overall, ddPCR shows superiority for clinical diagnosis of SARS-CoV-2 to reduce the false negative reports, which could be a powerful complement to the current standard RT-PCR. It also suggests that the current clinical practice that the convalescent after discharge continues to be quarantined for at least 2 weeks is completely necessary which can prevent potential viral transmission.

scholarly journals One-step nested RT-PCR for COVID-19 detection: A flexible, locally developed test for SARS-CoV2 nucleic acid detection

2020 ◽  
Vol 14 (07) ◽  
pp. 679-684 ◽  
Author(s):  
Carmen Meza-Robles ◽  
Carlos E Barajas-Saucedo ◽  
Daniel Tiburcio-Jimenez ◽  
Karen A Mokay-Ramírez ◽  
Valery Melnikov ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Positive Control ◽  
Nucleic Acid Detection ◽  
Rt Pcr ◽  
Current Standard ◽  
One Step ◽  
Species Specific ◽  
Bat Coronavirus ◽  
Nucleotide Region

Introduction: Due to the coronavirus pandemic, identifying the infected individuals has become key to limiting its spread. Virus nucleic acid real-time RT-PCR testing has become the current standard diagnostic method but high demand could lead to shortages. Therefore, we propose a detection strategy using a one-step nested RT-PCR. Methodology: The nucleotide region in the ORF1ab gene that has the greatest differences between the human coronavirus and the bat coronavirus was selected. Primers were designed after that sequence. All diagnostic primers are species-specific since the 3´ end of the sequence differs from that of other species. A primer set also creates a synthetic positive control. Amplified products were seen in a 2.5% agarose gel, as well as in an SYBR Green-Based Real-Time RT-PCR. Results: Amplification was achieved for the positive control and specific regions in both techniques. Conclusions: This new technique is flexible and easy to implement. It does not require a real-time thermocycler and can be interpreted in agarose gels, as well as adapted to quantify the viral genome. It has the advantage that if the coronavirus mutates in one of the key amplification nucleotides, at least one pair can still amplify, thanks to the four diagnostic primers.

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