scholarly journals Patient DNA cross-reactivity of the CDC SARS-CoV-2 extraction control leads to an inherent potential for false negative results

2020 ◽  
Author(s):  
Adam P. Rosebrock
Keyword(s):  
Reverse Transcription ◽  
False Negative ◽  
Rna Extraction ◽  
Cross Reactivity ◽  
Sample Collection ◽  
Negative Results ◽  
False Negative Results ◽  
Important Diagnostic Tool ◽  
Control And Prevention ◽  
Careful Handling

AbstractTesting for RNA viruses such as SARS-CoV-2 requires careful handling of inherently labile RNA during sample collection, clinical processing, and molecular analysis. Tests must include fail-safe controls that affirmatively report the presence of intact RNA and demonstrate success of all steps of the assay. A result of “no virus signal” is insufficient for clinical interpretation: controls must also say “The reaction worked as intended and would have found virus if present.” Unfortunately, a widely used test specified by the US Centers for Disease Control and Prevention (CDC) incorporates a control that does not perform as intended and claimed. Detecting SARS-CoV-2 with this assay requires both intact RNA and successful reverse transcription. The CDC-specified control does not require either of these, due to its inability to differentiate human genomic DNA from reverse-transcribed RNA. Patient DNA is copurified from nasopharyngeal swabs during clinically-approved RNA extraction and is sufficient to return an “extraction control success” signal using the CDC design. As such, this assay fails-unsafe: truly positive patient samples return a false-negative result of “no virus detected, control succeeded” following any of several readily-encountered mishaps. This problem affects tens-of-millions of patients worth of shipped assays, but many of these flawed reagents have not yet been used. There is an opportunity to improve this important diagnostic tool. As demonstrated here, a re-designed transcript-specific control correctly monitors sample collection, extraction, reverse transcription, and qPCR detection. This approach can be rapidly implemented and will help reduce truly positive patients from being incorrectly given the all-clear.One Sentence SummaryA widely-used COVID-19 diagnostic is mis-designed and generates false-negative results, dangerously confusing “No” with “Don’t know” – but it’s fixable

scholarly journals Five days of fever and myocardial inflammation

2021 ◽  
Vol 9 ◽  
pp. 2050313X2110508
Author(s):  
Keli D Coleman ◽  
Paul Benz ◽  
Nirzar S Parikh ◽  
Danny G Thomas ◽  
David Segar ◽  
...  
Keyword(s):  
False Negative ◽  
Clinical Signs ◽  
Myocardial Inflammation ◽  
Negative Results ◽  
Pediatric Illness ◽  
Multisystem Involvement ◽  
False Negative Results ◽  
Control And Prevention ◽  
Evidence Based Guidelines ◽  
Inflammatory Syndrome

Multisystem inflammatory syndrome in children is an emerging pediatric illness associated with severe acute respiratory syndrome coronavirus 2 infection. The syndrome is rare, and evidence-based guidelines are lacking. This report reviews a patient who presented for medical care multiple times early in the course of his illness, thus offering near-daily documentation of symptoms and laboratory abnormalities. The patient did not have thrombocytopenia, anemia, or myocardial inflammation until the fifth day of fever. These laboratory abnormalities coincided with the onset of rash, conjunctival injection, vomiting, and diarrhea: clinical signs that could serve as indicators for when to obtain blood tests. The timing of this patient’s onset of multisystem involvement suggests that testing for multisystem inflammatory syndrome in children after only 24 h of fever, as the Centers for Disease Control and Prevention recommends, may yield false-negative results. Testing for multisystem inflammatory syndrome in children after 4 days of fever may be more reliable.

scholarly journals Evaluation of symptomatic patient saliva as a sample type for the Abbott ID NOW COVID-19 assay

2020 ◽  
Author(s):  
Jeffrey A. SoRelle ◽  
Lenin Mahimainathan ◽  
Clare McCormick-Baw ◽  
Dominick Cavuoti ◽  
Francesca Lee ◽  
...  
Keyword(s):  
False Negative ◽  
Symptomatic Patient ◽  
Sample Type ◽  
Sample Collection ◽  
Cycle Number ◽  
Negative Results ◽  
Percent Agreement ◽  
False Negative Results ◽  
Nasal Swabs ◽  
Comparable Performance

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has presented significant challenges for laboratories including supply chain limitations with restricted access to reagents and sample collection materials (i.e. swabs, viral transport media (VTM)) for patients testing. Therefore, saliva has been evaluated as an alternative specimen for COVID-19 diagnosis. comparable performance between dry nasal swabs (NS) and nasopharyngeal swabs (NPS) collected in VTM has been observed with the ID NOW for SARS-CoV-2; the majority of false-negative results occur with higher cycle number (CN) or cycle threshold (Ct) values suggesting low viral load in these specimens. We performed clinical validation of saliva specimens on the ID NOW molecular platform to detect SARS-CoV-2. Saliva was compared to nasopharyngeal swabs tested on the ID NOW and the Cepheid molecular assay. We also performed stability studies of saliva samples over 5 days. A total of 96 saliva samples and 64 paired NPS were analyzed. We observed 78% (18/23) positive percent agreement (PPA) and 100% (44/44) negative percent agreement (NPA) between matched saliva and nasopharyngeal specimens performed on ID NOW. We found 83% (19/23) PPA and 100% NPA (25/25) between saliva run on the ID NOW and Cepheid assay. Six saliva specimens positive for SARS-CoV-2 were continuously positive for five days when stored at room temperature. Therefore, we propose further investigation of saliva as an alternative sample type for testing symptomatic patients with ID NOW as a promising method to address COVID-19 testing.

scholarly journals The value of repeat patient testing for SARS-CoV-2: real-world experience during the first wave

2021 ◽  
Vol 3 (7) ◽  
Author(s):  
Alex Zhu ◽  
Margaret Creagh ◽  
Chao Qi ◽  
Shannon Galvin ◽  
Maureen Bolon ◽  
...  
Keyword(s):  
False Negative ◽  
Laboratory Diagnosis ◽  
Nucleic Acid Amplification ◽  
Repeat Testing ◽  
The Third ◽  
Reverse Transcription Pcr ◽  
Negative Results ◽  
False Negative Results ◽  
Number Of Patients ◽  
Control And Prevention

Introduction. Reports of false-negative quantitative reverse transcription PCR (RT-qPCR) results from patients with high clinical suspension for coronavirus disease 2019 (COVID-19), suggested that a negative result produced by a nucleic acid amplification assays (NAAs) did not always exclude the possibility of COVID-19 infection. Repeat testing has been used by clinicians as a strategy in an to attempt to improve laboratory diagnosis of COVID-19 and overcome false-negative results in particular. Aim. To investigate whether repeat testing is helpful for overcoming false-negative results. Methods. We retrospectively reviewed our experience with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing, focusing on the yield of repeat patient testing for improving SARS-CoV-2 detection by NAA. Results. We found that the yield from using repeat testing to identify false-negative patients was low. When the first test produced a negative result, only 6 % of patients tested positive by the second test. The yield decreased to 1.7 and then 0 % after the third and fourth tests, respectively. When comparing the results produced by three assays, the Centers for Disease Control and Prevention (CDC) SARS CoV-2 RT-qPCR panel, Xpert Xpress CoV-2 and ID NOW COVID-19, the ID NOW assay was associated with the highest number of patients who tested negative initially but positive on repeat testing. The CDC SARS CoV-2 RT-qPCR panel produced the highest number of indeterminate results. Repeat testing resolved more than 90 % of indeterminate/invalid results. Conclusions. The yield from using repeat testing to identify false-negative patients was low. Repeat testing was best used for resolving indeterminate/invalid results.

scholarly journals False-negative Molecular Diagnosis of SARS-CoV-2 in Samples with Amplification Inhibitors

2020 ◽  
Author(s):  
Marcelo Fruehwirth ◽  
Açucena Veleh Rivas ◽  
Andressa Faria Rahyn Fitz ◽  
Aline Cristiane Cechinel Assing Batista ◽  
Cleypson Vinicius Silveira ◽  
...  
Keyword(s):  
Internal Control ◽  
False Negative ◽  
Rna Extraction ◽  
N Gene ◽  
Gold Standard Method ◽  
Negative Results ◽  
False Negative Results ◽  
Average Variation ◽  
Wrong Diagnosis ◽  
Inhibitor Compounds

Although rRT-PCR is the gold standard method for SARS-CoV-2 detection, some factors, such as amplification inhibitors presence, lead to false-negative results. Here we describe differences between rRT-PCR results for SARS-CoV-2 infection in normal and diluted samples, simulating the need for dilution due to amplification inhibitors presence. Viral RNA extraction of nasopharyngeal swabs samples from 20 patients previously detected as 'Negative' and 21 patients detected as 'Positive' for SARS-CoV-2 was realized with the EasyExtract DNA-RNA (Interprise®). rRT-PCR was realized with OneStep/COVID-19 (IBMP) kit with normal and diluted (80µl of H₂O RNAse free) samples, totaling 82 tests. The results indicate that there is an average variation (ɑ < 0.05) delaying Cq between the amplification results of internal control (IC), N Gene (NG), and ORF-1ab (OF) of 1.811 Cq, 3.840 Cq, and 3.842 Cq, respectively. The extraction kit does not completely purify the inhibitor compounds, therefore non-amplification by inhibitors may occur. In this study, we obtained a 19.04% false-negative diagnosis after sample dilution, and this process reduces the efficiency of rRT-PCR to 29.80% for detecting SARS-CoV-2. Knowing the rRT-PCR standards of diluted samples can help in the identification of false-negative cases, and consequently avoid a wrong diagnosis.

scholarly journals CovidArray: A Microarray-Based Assay with High Sensitivity for the Detection of Sars-Cov-2 in Nasopharyngeal Swabs

Sensors ◽  
2021 ◽  
Vol 21 (7) ◽  
pp. 2490
Author(s):  
Francesco Damin ◽  
Silvia Galbiati ◽  
Stella Gagliardi ◽  
Cristina Cereda ◽  
Francesca Dragoni ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Solid Phase ◽  
Quantitative Polymerase Chain Reaction ◽  
False Negative ◽  
Rna Extraction ◽  
High Sensitivity ◽  
Great Sensitivity ◽  
Viral Rna ◽  
Analytical Sensitivity ◽  
Negative Results

A new coronavirus (SARS-CoV-2) caused the current coronavirus disease (Covid-19) epidemic. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used as the gold standard for clinical detection of SARS-CoV-2. Under ideal conditions, RT-qPCR Covid-19 assays have analytical sensitivity and specificity greater than 95%. However, when the sample panel is enlarged including asymptomatic individuals, the sensitivity decreases and false negatives are reported. Moreover, RT-qPCR requires up to 3–6 h with most of the time involved in RNA extraction from swab samples. We introduce CovidArray, a microarray-based assay, to detect SARS-CoV-2 markers N1 and N2 in the nasopharyngeal swabs. The method is based on solid-phase hybridization of fluorescently-labeled amplicons upon RNA extraction and reverse transcription. This approach combines the physical-optical properties of the silicon substrate with the surface chemistry used to coat the substrate to obtain a diagnostic tool of great sensitivity. Furthermore, we used an innovative approach, RNAGEM, to extract and purify viral RNA in less than 15 min. We correctly assigned 12 nasopharyngeal swabs, previously analyzed by RT-qPCR. Thanks to the CovidArray sensitivity we were able to identify a false-negative sample. CovidArray is the first DNA microarray-based assay to detect viral genes in the swabs. Its high sensitivity and the innovative viral RNA extraction by RNAGEM allows the reduction of both the amount of false-negative results and the total analysis time to about 2 h.

scholarly journals Identification of Novel Mutations in the N Gene of SARS-CoV-2 That Adversely Affect the Detection of the Virus by Reverse Transcription-Quantitative PCR

2021 ◽  
Author(s):  
Rashedul Hasan ◽  
Mohammad Enayet Hossain ◽  
Mojnu Miah ◽  
Md Mahmudul Hasan ◽  
Mustafizur Rahman ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Reverse Transcription ◽  
Binding Sites ◽  
False Negative ◽  
N Gene ◽  
Novel Mutations ◽  
Viral Spread ◽  
Negative Results ◽  
False Negative Results ◽  
Reverse Transcription Quantitative Pcr

Accurate and timely diagnosis of SARS-CoV-2 is a critical step toward controlling the viral spread, since it facilitates the identification and isolation of infected individuals. Mutations in the primer-/probe-binding sites may lead to false-negative results.

scholarly journals False-Negative Molecular Diagnosis of SARS-CoV-2 in Samples with Amplification Inhibitors

2020 ◽  
Author(s):  
Marcelo Fruehwirth ◽  
Açucena Veleh Rivas ◽  
Andressa Faria Rahyn Fitz ◽  
Aline Cristiane Cechinel Assing Batista ◽  
Cleypson Vinicius Silveira ◽  
...  
Keyword(s):  
Internal Control ◽  
False Negative ◽  
Rna Extraction ◽  
N Gene ◽  
Gold Standard Method ◽  
Negative Results ◽  
False Negative Results ◽  
Average Variation ◽  
Wrong Diagnosis ◽  
Inhibitor Compounds

Although rRT-PCR is the gold standard method for SARS-CoV-2 detection, some factors, such as amplification inhibitors presence, lead to false-negative results. Here we describe differences between rRT-PCR results for SARS-CoV-2 infection in normal and diluted samples, simulating the need for dilution due to amplification inhibitors presence. Viral RNA extraction of nasopharyngeal swabs samples from 20 patients previously detected as 'Negative' and 21 patients detected as 'Positive' for SARS-CoV-2 was realized with the EasyExtract DNA-RNA (Interprise®) for extraction. rRT-PCR was realized with OneStep/COVID-19 (IBMP) kit with normal and diluted (80µl of H₂O RNAse free) samples, totaling 82 tests. The results indicate that there is an average variation (ɑ < 0.05) delaying Ct between the amplification results of internal control (IC), N Gene (NG), and ORF-1ab (OF) of 1.811Ct, 3.840Ct, and 3.842Ct, respectively. The extraction kit does not completely purify the inhibitor compounds, therefore non-amplification by inhibitors may occur. In this study, we obtained a 19.04% false-negative diagnosis after sample dilution, and this process reduces the efficiency of rRT-PCR to 29.8% for detecting SARS-CoV-2. Knowing the rRT-PCR standards of diluted samples can help in the identification of false-negative cases, and consequently avoid a wrong diagnosis.

scholarly journals Molecular detection of SARS-CoV-2 using a reagent-free approach

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0243266
Author(s):  
Ronan Calvez ◽  
Andrew Taylor ◽  
Leonides Calvo-Bado ◽  
Donald Fraser ◽  
Colin G. Fink
Keyword(s):  
Internal Control ◽  
Molecular Detection ◽  
False Negative ◽  
Rna Extraction ◽  
Processing Method ◽  
Heat Processing ◽  
Negative Results ◽  
Treatment Conditions ◽  
False Negative Results ◽  
Different Temperatures

Shortage of reagents and consumables required for the extraction and molecular detection of SARS-CoV-2 RNA in respiratory samples has led many laboratories to investigate alternative approaches for sample preparation. Many groups recently presented results using heat processing method of respiratory samples prior to RT-qPCR as an economical method enabling an extremely fast streamlining of the processes at virtually no cost. Here, we present our results using this method and highlight some major pitfalls that diagnostics laboratories should be aware of before proceeding with this methodology. We first investigated various treatments using different temperatures, incubation times and sample volumes to optimise the heat treatment conditions. Although the initial data confirmed results published elsewhere, further investigations revealed unexpected inhibitory properties of some commonly used universal transport media (UTMs) on some commercially available RT-qPCR mixes, leading to a risk of reporting false-negative results. This emphasises the critical importance of a thorough validation process to determine the most suitable reagents to use depending on the sample types to be tested. In conclusion, a heat processing method is effective with very consistent Ct values and a sensitivity of 96.2% when compared to a conventional RNA extraction method. It is also critical to include an internal control to check each sample for potential inhibition.

scholarly journals 689. Nasal versus Nasopharyngeal Sample Collection for Diagnostic Nucleic Acid Amplification Testing for Influenza A, Influenza B, and Respiratory Syncytial Viruses

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S398-S398
Author(s):  
Grace A Schaack ◽  
Brittney Jung-Hynes ◽  
Derrick Chen
Keyword(s):  
Nucleic Acid ◽  
Influenza A ◽  
False Negative ◽  
Influenza Viruses ◽  
Viral Rna ◽  
Nucleic Acid Amplification ◽  
Influenza B ◽  
Sample Collection ◽  
Negative Results ◽  
False Negative Results

Abstract Background Nucleic acid amplification testing (NAAT) for influenza A virus (IAV), influenza B virus (IBV), and respiratory syncytial virus (RSV) is a standard component of diagnosis of infections with these pathogens. At our institution, current standard of practice is to collect nasopharyngeal (NP) samples for such NAAT. In an effort to provide clinicians and patients a simpler, more comfortable sample collection option, we evaluated the use of nasal samples for NAAT, compared to NP samples. Methods Both nasal and NP specimens were collected from each of 58 patients seen in our emergency department (January – March 2020). NP samples were collected using minitip FLOQswabs; nasal samples were collected using regular or minitip FLOQswabs. Nasal and NP samples were processed using the same protocol and tested for influenza viruses and RSV using the Cepheid GeneXpert (Xpress Flu/RSV) platform. Results In total, 20 NP samples tested negative for virus and 38 tested positive (16 IAV-positive, 14 IBV-positive, 8 RSV-positive). There were 3 cases (5% of total cases) in which qualitative (positive/negative) results from the corresponding nasal samples were not in agreement with results derived from NP samples. These were considered false-negative results; one such discrepancy was resolved upon re-testing the same samples. Overall positive percent agreement between nasal- and NP-derived results was 92% (35/38), and negative percent agreement was 100% (20/20). Among samples testing positive for virus by both NP and nasal sampling methods, we found that the average cycle threshold (Ct) value for IAV detection was 5.1 cycles (n = 16, SEM = 0.83) higher for nasal samples than for NP samples. The average Ct for IBV detection was 3.3 cycles (n = 12, SEM = 1.87) higher for nasal than for NP samples. The average Ct for RSV detection was 1.9 cycles (n = 7, SEM = 1.57) higher for nasal than for NP samples. Conclusion These results suggest that recovery of viral RNA from nasal samples is lower than that from nasopharyngeal samples, particularly for influenza viruses. This decreased detection of viral RNA from nasal samples may explain the false-negative results seen in our discrepant cases. These data suggest that a decrease in recovery of viral RNA by nasal sampling may translate to decreased diagnostic accuracy. Disclosures All Authors: No reported disclosures

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