scholarly journals Combined RT-qPCR and Pyrosequencing of a SARS-CoV-2 Spike Glycoprotein Polybasic Cleavage Motif Uncovers Rare Pediatric COVID-19 Spectrum Diseases of Unusual Presentation

2020 ◽  
Author(s):  
Patrick Philipp Weil ◽  
Jacqueline Hentschel ◽  
Frank Schult ◽  
Anton Pembaur ◽  
Beniam Ghebremedhin ◽  
...  
Keyword(s):  
Best Practice ◽  
Quantitative Polymerase Chain Reaction ◽  
False Negative ◽  
Detection Limits ◽  
Epidemiologic Studies ◽  
Viral Loads ◽  
S Gene ◽  
False Positive Diagnosis ◽  
Containment Measures ◽  
Pyrosequencing Method

AbstractBackgroundSurveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is essential for the global containment measures with regard to the ongoing pandemic. Diagnostic gold standard is currently reverse transcription of the (+)RNA genome and subgenomic RNAs and subsequent quantitative polymerase chain reaction (RT-qPCR) from nasopharyngeal swabs or bronchoalveolar lavages. In order to further improve the diagnostic accuracy, particularly for the reliable discrimination between negative and false-negative specimens, we propose the combination of the RT-qPCR workflow with subsequent pyrosequencing of a S-gene amplicon. This extension might add important value mainly in cases with low SARS-CoV-2 load, where RT-qPCR alone can deliver conflicting results.sResultsWe successfully established a combined RT-qPCR and S-gene pyrosequencing method which can be optionally exploited after routine diagnostics or for epidemiologic studies. This allows a more reliable interpretation of conflicting RT-qPCR results in specimens with relatively low viral loads and close to the detection limits of qPCR (CT values >30). After laboratory implementation and characterization of a best practice protocol we tested the combined method in a large pediatric cohort from two German medical centers (n=769). Pyrosequencing after RT-qPCR enabled us to uncover 6 previously unrecognized cases of pediatric SARS-CoV-2 associated diseases, partially exhibiting unusual and heterogeneous presentation. Moreover, it is notable that in the course of RT-qPCR/pyrosequencing method establishment we did not observe any case of false-positive diagnosis when confirmed SARS-CoV-2-positive specimens were used from foregoing routine testing.ConclusionsThe proposed protocol allows a specific and sensitive detection of SARS-CoV-2 close to the detection limits of RT-qPCR. Combined RT-qPCR/pyrosequencing does not negatively affect preceding RT-qPCR pipeline in SARS-CoV-2 diagnostics and can be optionally applied in routine to inspect conflicting RT-qPCR results.

scholarly journals Combined RT-qPCR and pyrosequencing of a Spike glycoprotein polybasic cleavage motif can uncover pediatric SARS-CoV-2 infections associated with heterogeneous presentation

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Patrick Philipp Weil ◽  
Jacqueline Hentschel ◽  
Frank Schult ◽  
Anton Pembaur ◽  
Beniam Ghebremedhin ◽  
...  
Keyword(s):  
Single Case ◽  
Quantitative Polymerase Chain Reaction ◽  
False Negative ◽  
Detection Limits ◽  
Antibody Testing ◽  
Viral Loads ◽  
S Gene ◽  
Containment Measures ◽  
Reliable Interpretation ◽  
Cleavage Motif

Abstract Background Reverse transcription of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (+)RNA genome and subgenomic RNAs (sgRNAs) and subsequent quantitative polymerase chain reaction (RT-qPCR) is the reliable diagnostic gold standard for COVID-19 diagnosis and the identification of potential spreaders. Apart from clinical relevance and containment, for specific questions, it might be of interest to (re)investigate cases with low SARS-CoV-2 load, where RT-qPCR alone can deliver conflicting results, even though these cases might neither be clinically relevant nor significant for containment measures, because they might probably not be infectious. In order to expand the diagnostic bandwidth for non-routine questions, particularly for the reliable discrimination between negative and false-negative specimens associated with high CT values, we combined the RT-qPCR workflow with subsequent pyrosequencing of a S-gene amplicon. This expansion can help to confirm SARS-CoV-2 infections without the demand of confirmative antibody testing, which requires to summon patients again for blood sampling few to several weeks after symptom onset. Results We successfully established a combined RT-qPCR and S-gene pyrosequencing method which can be optionally exploited after routine diagnostics. This allows a reliable interpretation of RT-qPCR results in specimens with relatively low viral loads and close to the detection limits of qPCR. After laboratory implementation, we tested the combined method in a large pediatric cohort from two German medical centers (n=769). Pyrosequencing after RT-qPCR enabled us to uncover 5 previously unrecognized cases of pediatric SARS-CoV-2-associated diseases, mainly exhibiting mild and heterogeneous presentation—apart from a single case of multisystem inflammatory syndrome in children (MIS-C) associated with SARS-CoV-2, who was hospitalized in the course of the study. Conclusions The proposed protocol allows a specific and sensitive confirmation of SARS-CoV-2 infections close to the detection limits of RT-qPCR. The tested biotinylated primers do not negatively affect the RT-qPCR pipeline and thus can be optionally applied to enable deeper inspection of RT-qPCR results by subsequent pyrosequencing. Moreover, due to the incremental transmission of SARS-CoV-2 variants of concern, we note that the used strategy can uncover (Spike) P681H allowing the pre-selection of SARS-CoV-2 B.1.1.7 candidate specimens for deep sequencing.

scholarly journals Estimating epidemiologic dynamics from cross-sectional viral load distributions

Science ◽  
2021 ◽  
pp. eabh0635
Author(s):  
James A. Hay ◽  
Lee Kennedy-Shaffer ◽  
Sanjat Kanjilal ◽  
Niall J. Lennon ◽  
Stacey B. Gabriel ◽  
...  
Keyword(s):  
Population Distribution ◽  
Quantitative Polymerase Chain Reaction ◽  
Cross Sectional ◽  
Outbreak Management ◽  
Viral Loads ◽  
Time Estimates ◽  
Load Distributions ◽  
Random Samples ◽  
Combining Data ◽  
Polymerase Chain

Estimating an epidemic’s trajectory is crucial for developing public health responses to infectious diseases, but case data used for such estimation are confounded by variable testing practices. We show that the population distribution of viral loads observed under random or symptom-based surveillance, in the form of cycle threshold (Ct) values obtained from reverse-transcription quantitative polymerase chain reaction testing, changes during an epidemic. Thus, Ct values from even limited numbers of random samples can provide improved estimates of an epidemic’s trajectory. Combining data from multiple such samples improves the precision and robustness of such estimation. We apply our methods to Ct values from surveillance conducted during the SARS-CoV-2 pandemic in a variety of settings and offer alternative approaches for real-time estimates of epidemic trajectories for outbreak management and response.

scholarly journals Evaluation of COVID-19 RT-qPCR Test in Multi sample Pools

2020 ◽  
Vol 71 (16) ◽  
pp. 2073-2078 ◽  
Author(s):  
Idan Yelin ◽  
Noga Aharony ◽  
Einat Shaer Tamar ◽  
Amir Argoetti ◽  
Esther Messer ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
False Negative ◽  
Rna Extraction ◽  
False Negative Rate ◽  
Positive Sample ◽  
Recent Emergence ◽  
Single Positive ◽  
Polymerase Chain ◽  
Pool Test ◽  
Current Screening

Abstract Background The recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to a current pandemic of unprecedented scale. Although diagnostic tests are fundamental to the ability to detect and respond, overwhelmed healthcare systems are already experiencing shortages of reagents associated with this test, calling for a lean immediately applicable protocol. Methods RNA extracts of positive samples were tested for the presence of SARS-CoV-2 using reverse transcription quantitative polymerase chain reaction, alone or in pools of different sizes (2-, 4-, 8-, 16-, 32-, and 64-sample pools) with negative samples. Transport media of additional 3 positive samples were also tested when mixed with transport media of negative samples in pools of 8. Results A single positive sample can be detected in pools of up to 32 samples, using the standard kits and protocols, with an estimated false negative rate of 10%. Detection of positive samples diluted in even up to 64 samples may also be attainable, although this may require additional amplification cycles. Single positive samples can be detected when pooling either after or prior to RNA extraction. Conclusions As it uses the standard protocols, reagents, and equipment, this pooling method can be applied immediately in current clinical testing laboratories. We hope that such implementation of a pool test for coronavirus disease 2019 would allow expanding current screening capacities, thereby enabling the expansion of detection in the community, as well as in close organic groups, such as hospital departments, army units, or factory shifts.

scholarly journals Accumulation of Azelaic Acid in Xylella fastidiosa-Infected Olive Trees: A Mobile Metabolite for Health Screening

2019 ◽  
Vol 109 (2) ◽  
pp. 318-325 ◽  
Author(s):  
Francesca Nicolì ◽  
Carmine Negro ◽  
Eliana Nutricati ◽  
Marzia Vergine ◽  
Alessio Aprile ◽  
...  
Keyword(s):  
Azelaic Acid ◽  
Large Scale ◽  
Systemic Acquired Resistance ◽  
Xylella Fastidiosa ◽  
Quantitative Polymerase Chain Reaction ◽  
False Negative ◽  
Screening Method ◽  
Expression Level ◽  
Specific Domain ◽  
Olive Trees

Monitoring Xylella fastidiosa is critical for eradicating or at least containing this harmful pathogen. New low-cost and rapid methods for early detection capability are very much needed. Metabolomics may play a key role in diagnosis; in fact, mobile metabolites could avoid errors in sampling due to erratically distributed pathogens. Of the various different mobile signals, we studied dicarboxylic azelaic acid (AzA) which is a key molecule for biotic stress plant response but has not yet been associated with pathogens in olive trees. We found that infected Olea europaea L. plants of cultivars Cellina di Nardò (susceptible to X. fastidiosa) and Leccino (resistant to the pathogen) showed an increase in AzA accumulation in leaf petioles and in sprigs by approximately seven- and sixfold, respectively, compared with plants negative to X. fastidiosa or affected by other pathogens. No statistically significant variation was found between the X. fastidiosa population level and the amount of AzA in either of the plant tissues, suggesting that AzA accumulation was almost independent of the amount of pathogen in the sample. Furthermore, the association of AzA with X. fastidiosa seemed to be reliable for samples judged as potentially false-negative by quantitative polymerase chain reaction (cycle threshold [Ct] > 33), considering both the absolute value of AzA concentration and the values normalized on negative samples, which diverged significantly from control plants. The accumulation of AzA in infected plants was partially supported by the differential expression of two genes (named OeLTP1 and OeLTP2) encoding lipid transport proteins (LTPs), which shared a specific domain with the LTPs involved in AzA activity in systemic acquired resistance in other plant species. The expression level of OeLTP1 and OeLTP2 in petiole samples showed significant upregulation in samples positive to X. fastidiosa of both cultivars, with higher expression levels in positive samples of Cellina di Nardò compared with Leccino, whereas the two transcripts had a low expression level (Ct > 40) in negative samples of the susceptible cultivar. Although the results derived from the quantification of AzA cannot confirm the presence of the erratically distributed X. fastidiosa, which can be definitively assessed by traditional methods, we believe they represent a fast and cheap screening method for large-scale monitoring.

scholarly journals Microfluidic Nano-Scale qPCR Enables Ultra-Sensitive and Quantitative Detection of SARS-CoV-2

Processes ◽  
2020 ◽  
Vol 8 (11) ◽  
pp. 1425
Author(s):  
Xin Xie ◽  
Tamara Gjorgjieva ◽  
Zaynoun Attieh ◽  
Mame Massar Dieng ◽  
Marc Arnoux ◽  
...  
Keyword(s):  
Viral Infections ◽  
Limit Of Detection ◽  
False Negative ◽  
False Negative Rate ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
Viral Loads ◽  
Nano Scale ◽  
Negative Rate

A major challenge in controlling the COVID-19 pandemic is the high false-negative rate of the commonly used RT-PCR methods for SARS-CoV-2 detection in clinical samples. Accurate detection is particularly challenging in samples with low viral loads that are below the limit of detection (LoD) of standard one- or two-step RT-PCR methods. In this study, we implemented a three-step approach for SARS-CoV-2 detection and quantification that employs reverse transcription, targeted cDNA preamplification, and nano-scale qPCR based on a commercially available microfluidic chip. Using SARS-CoV-2 synthetic RNA and plasmid controls, we demonstrate that the addition of a preamplification step enhances the LoD of this microfluidic RT-qPCR by 1000-fold, enabling detection below 1 copy/µL. We applied this method to analyze 182 clinical NP swab samples previously diagnosed using a standard RT-qPCR protocol (91 positive, 91 negative) and demonstrate reproducible and quantitative detection of SARS-CoV-2 over five orders of magnitude (<1 to 106 viral copies/µL). Crucially, we detect SARS-CoV-2 with relatively low viral load estimates (<1 to 40 viral copies/µL) in 17 samples with negative clinical diagnosis, indicating a potential false-negative rate of 18.7% by clinical diagnostic procedures. In summary, this three-step nano-scale RT-qPCR method can robustly detect SARS-CoV-2 in samples with relatively low viral loads (<1 viral copy/µL) and has the potential to reduce the false-negative rate of standard RT-PCR-based diagnostic tests for SARS-CoV-2 and other viral infections.

scholarly journals Quantitative polymerase chain reaction for human herpesvirus diagnosis and measurement of Epstein–Barr virus burden in posttransplant lymphoproliferative disorder

1997 ◽  
Vol 43 (10) ◽  
pp. 1843-1849 ◽  
Author(s):  
Xin Bai ◽  
Gregory Hosler ◽  
Beverly Barton Rogers ◽  
D Brian Dawson ◽  
Richard H Scheuermann
Keyword(s):  
Quantitative Pcr ◽  
Lymphoproliferative Disorder ◽  
Quantitative Polymerase Chain Reaction ◽  
False Negative ◽  
Internal Standard ◽  
Human Herpesvirus ◽  
Transplant Patients ◽  
Viral Burden ◽  
Pcr Method ◽  
Human Herpesviruses

Abstract Human herpesviruses can cause acute diseases such as chicken pox or mononucleosis, but also may reactivate during immunosuppression and result in severe or life-threatening illnesses such as shingles or lymphoproliferative disorders. We report the development and validation of a quantitative PCR method to measure viral burden for all eight human herpesviruses (HSV1, HSV2, VZV, EBV, CMV, HHV6, HHV7, and KSHV) in patients’ samples. The method uses an internal standard that is coamplified with the viral target, allowing quantification of viral genomes in absolute terms (e.g., viral targets/mL of blood) and ruling out false-negative results. We demonstrate that transplant patients with lymphoproliferative disorder carry an EBV viral burden 3 logs higher than nontransplant patients. EBV titers in transplant patients without a lymphoproliferative disorder are between these values. This quantitative PCR method may aid in differentiating clinically significant vs latent viral burden in immunosuppressed patients.

Automated Method for Determining Serum Thiocyanate, to Distinguish Smokers from Nonsmokers

1974 ◽  
Vol 20 (10) ◽  
pp. 1344-1348 ◽  
Author(s):  
William C Butts ◽  
Martha Kuehneman ◽  
Graham M Widdowson
Keyword(s):  
False Negative ◽  
Epidemiologic Studies ◽  
Ferric Ions ◽  
Colored Complex ◽  
Chemical Test ◽  
Negative Results ◽  
Automated Method ◽  
Serum Thiocyanate ◽  
False Negative Results ◽  
Two Populations

Abstract A chemical test to distinguish smokers and nonsmokers is important in many epidemiologic studies. We have developed an automated method for determining serum thiocyanate by use of the reaction between thiocyanate and ferric ions to form a colored complex. Our data show that this method distinguishes cigarette smokers and nonsmokers: of 197 healthy individuals studied, 1.8% false-positive and 6.7% false-negative results were obtained when 85 µmol of SCN- per liter was the critical value used to distinguish the two populations.

scholarly journals A Naturally Transmitted Epitheliotropic Polyomavirus Pathogenic in Immunodeficient Rats: Characterization, Transmission, and Preliminary Epidemiologic Studies

2017 ◽  
Vol 45 (5) ◽  
pp. 593-603 ◽  
Author(s):  
Cynthia Besch-Williford ◽  
Patricia Pesavento ◽  
Shari Hamilton ◽  
Beth Bauer ◽  
Beatrix Kapusinszky ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Prostate Gland ◽  
Interleukin 2 ◽  
Epidemiologic Studies ◽  
Metagenomic Sequencing ◽  
Serum Samples ◽  
Diagnostic Assays ◽  
Wu Polyomavirus ◽  
Washington University

We report the identification, pathogenesis, and transmission of a novel polyomavirus in severe combined immunodeficient F344 rats with null Prkdc and interleukin 2 receptor gamma genes. Infected rats experienced weight loss, decreased fecundity, and mortality. Large basophilic intranuclear inclusions were observed in epithelium of the respiratory tract, salivary and lacrimal glands, uterus, and prostate gland. Unbiased viral metagenomic sequencing of lesioned tissues identified a novel polyomavirus, provisionally named Rattus norvegicus polyomavirus 2 (RatPyV2), which clustered with Washington University (WU) polyomavirus in the Wuki clade of the Betapolyomavirus genus. In situ hybridization analyses and quantitative polymerase chain reaction (PCR) results demonstrated viral nucleic acids in epithelium of respiratory, glandular, and reproductive tissues. Polyomaviral disease was reproduced in Foxn1rnu nude rats cohoused with infected rats or experimentally inoculated with virus. After development of RatPyV2-specific diagnostic assays, a survey of immune-competent rats from North American research institutions revealed detection of RatPyV2 in 7 of 1,000 fecal samples by PCR and anti-RatPyV2 antibodies in 480 of 1,500 serum samples. These findings suggest widespread infection in laboratory rat populations, which may have profound implications for established models of respiratory injury. Additionally, RatPyV2 infection studies may provide an important system to investigate the pathogenesis of WU polyomavirus diseases of man.

Exploring 'best practice' for nucleic acid detection of Neisseria gonorrhoeae

Sexual Health ◽  
2008 ◽  
Vol 5 (1) ◽  
pp. 17 ◽  
Author(s):  
David M. Whiley ◽  
Suzanne M. Garland ◽  
Geoffrey Harnett ◽  
Gary Lum ◽  
David W. Smith ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Neisseria Gonorrhoeae ◽  
Best Practice ◽  
Current Knowledge ◽  
False Negative ◽  
High Sensitivity ◽  
Nucleic Acid Detection ◽  
Predictive Values ◽  
Neisseria Species ◽  
Negative Results

Nucleic acid detection tests (NADT) have considerable benefits for the detection of Neisseria gonorrhoeae (GC), including high sensitivity across a range of specimen types and use under widely differing settings and conditions. However, sexual health practitioners and others who use data generated by NADT for GC should be aware of some important limitations of these tests. False-positive results caused by cross reaction with commensal Neisseria species have been observed in many assays, and have lead to unacceptably low positive-predictive values in some patient populations. Further, false-negative results can be caused by GC sequence variation, with some gonococci lacking certain NADT target sequences. This review examines the issues associated with gonococcal NADT and considers best practice for use of these assays based on current knowledge. We emphasise the need for supplementary testing and extensive assay validation, and suggest appropriate strategies for these requirements irrespective of the setting in which they are used. Further, we highlight the need to maintain culture-based testing for certain specimen sites as well as for antimicrobial resistance surveillance.

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