Potential pitfalls in analysing a SARS-CoV-2 RT-PCR assay and how to standardise data interpretation.
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SUBMITTER: Smith M
PROVIDER: S-EPMC9307283 | biostudies-literature |
REPOSITORIES: biostudies-literature
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| S-EPMC7845522 | biostudies-literature
Project description:BackgroundReverse transcription-polymerase chain reaction (RT-PCR) is an extremely common clinical method for detecting pathogens, particularly for emerging infectious diseases such as the new coronavirus disease (COVID-19). Currently, detection of the RNA from the novel coronavirus SARS-CoV-2 is the gold standard for establishing a COVID-19 diagnosis. This study evaluates the characteristic performance of the analytical system in a clinical laboratory.MethodsA commercial SARS-CoV-2 RNA RT-PCR Kit used in a clinical laboratory is assessed based on ISO 15189 verification requirements. A multiple real-time RT-PCR assay for the RdRP, N, and E genes in SARS-CoV-2 is verified.ResultsThe analytical system exhibits good analytical sensitivity (1000 copies/mL) and specificity (100%); however, the values of 86.7% and 100% for analytical accuracy deserved attention, compared with two other types of methods. Overall, the kit is potentially useful for SARS-CoV-2 diagnostic testing and meets the verification requirements.ConclusionCompliance with international standards, such as ISO 15189, is valuable for clinical laboratories and for improving laboratory medicine quality and safety. Normalization is essential for obtaining reliable results from the SARS-CoV-2 RNA RT-PCR assay. This study aims to develop an improved SARS-CoV-2 verification framework compared with traditional molecular diagnostic methods, given the urgency of implementing new assays in clinical laboratories.
| S-EPMC7435359 | biostudies-literature
Project description:We aimed to evaluate the LIAISON® SARS-CoV-2 antigen assay (DiaSorin), comparing its performance to real-time polymerase chain reaction (RT-PCR) for the detection of SARS-CoV-2 RNA. 182 (110 PCR-positive and 72 PCR-negative) nasopharyngeal swab samples were taken for the detection of SARS-CoV-2. RT-PCR and antigen assay were performed using the same material. The sensitivity and specificity of the antigen assay were calculated for different cut-offs, with RT-PCR serving as the reference method. Stored clinical samples that were positive for other respiratory viruses were tested to evaluate cross-reactivity. One third (33/110, 30%) were falsely classified as negative, while no false positives were found using the 200 TCID50/mL cut-off for the SARS-CoV-2 antigen as proposed by the manufacturer. This corresponded to a sensitivity of 70% (60-78%) and a specificity of 100% (94-100%). Lowering the cut-off for positivity of the antigen assay to 22.79 or 57.68 TCID50/mL increased the sensitivity of the method, reaching a sensitivity of 92% (85-96%) vs. 79% (70-86%) and a specificity of 81% (69-89%) vs. 99% (91-100%), respectively. The antigen assay reliably detected samples with high SARS-CoV-2 viral loads (≥106 copies SARS-CoV-2/mL), while it cannot differentiate between negative and low positive samples. Cross-reactivity toward other respiratory viruses was not detected.
| S-EPMC8228578 | biostudies-literature
Project description:Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is the gold standard recommended to test for acute SARS-CoV-2 infection. However, it generally requires expensive equipment such as RNA isolation instruments and real-time PCR thermal cyclers. As a pandemic, COVID-19 has spread indiscriminately, and many low resource settings and developing countries do not have the means for fast and accurate COVID-19 detection to control the outbreak. Additionally, long assay times, in part caused by slow sample preparation steps, have created a large backlog when testing patient samples suspected of COVID-19. With many PCR-based molecular assays including an extraction step, this can take a significant amount of time and labor, especially if the extraction is performed manually. Using COVID-19 clinical specimens, we have collected evidence that the RT-qPCR assay can feasibly be performed directly on patient sample material in virus transport medium (VTM) without an RNA extraction step, while still producing sensitive test results. If RNA extraction steps can be omitted without significantly affecting clinical sensitivity, the turn-around time of COVID-19 tests, and the backlog we currently experience can be reduced drastically. Furthermore, our data suggest that rapid RT-PCR can be implemented for sensitive and specific molecular diagnosis of COVID-19 in locations where sophisticated laboratory instruments are not available. Our USD 300 set up achieved rapid RT-PCR using thin-walled PCR tubes and a water bath setup using sous vide immersion heaters, a Raspberry Pi computer, and a single servo motor that can process up to 96 samples at a time. Using COVID-19 positive clinical specimens, we demonstrated that RT-PCR assays can be performed in as little as 12 min using untreated samples, heat-inactivated samples, or extracted RNA templates with our low-cost water bath setup. These findings can help rapid COVID-19 testing to become more accessible and attainable across the globe.
| S-EPMC7598596 | biostudies-literature
Project description:Coronavirus disease 2019 (COVID-19) is an infectious, acute respiratory disease caused mainly by person-to-person transmission of the coronavirus SARS-CoV-2. Its emergence has caused a world-wide acute health crisis, intensified by the challenge of reliably identifying individuals likely to transmit the disease. Diagnosis is hampered by the many unknowns surrounding this disease, including those relating to infectious viral burden. This uncertainty is exacerbated by disagreement surrounding the clinical relevance of molecular testing using reverse transcription quantitative PCR (RT-qPCR) for the presence of viral RNA, most often based on the reporting of quantification cycles (Cq), which is also termed the cycle threshold (Ct) or crossing point (Cp). Despite it being common knowledge that Cqs are relative values varying according to a wide range of different parameters, there have been efforts to use them as though they were absolute units, with Cqs below an arbitrarily determined value, deemed to signify a positive result and those above, a negative one. Our results investigated the effects of a range of common variables on Cq values. These data include a detailed analysis of the effect of different carrier molecules on RNA extraction. The impact of sample matrix of buccal swabs and saliva on RNA extraction efficiency was demonstrated in RT-qPCR and the impact of potentially inhibiting compounds in urine along with bile salts were investigated in RT-digital PCR (RT-dPCR). The latter studies were performed such that the impact on the RT step could be separated from the PCR step. In this way, the RT was shown to be more susceptible to inhibitors than the PCR. Together, these studies demonstrate that the consequent variability of test results makes subjective Cq cut-off values unsuitable for the identification of infectious individuals. We also discuss the importance of using reliable control materials for accurate quantification and highlight the substantial role played by dPCR as a method for their development.
| S-EPMC8387146 | biostudies-literature
Project description:BackgroundIndia bears the second largest burden of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. A multitude of reverse transcription polymerase chain reaction (RT-PCR) detection assays with disparate gene targets, including automated high-throughput platforms, are available. Varying concordance and interpretation of diagnostic results in this setting can result in significant reporting delays, leading to suboptimal disease management. This article reports the development of a novel ORF1a-based SARS-CoV-2 RT-PCR assay - Viroselect - that shows high concordance with conventional assays and the ability to resolve inconclusive results generated during the peak of the epidemic in Mumbai, India.MethodsA unique target region within SARS-CoV-2 ORF1a - the non-structural protein 3 (nsp3) region - was used to design and develop the assay. This hypervariable region (1923-3956) between SARS-CoV-2, SARS-CoV-1 and Middle East respiratory syndrome coronavirus was utilized to design the primers and probes for the RT-PCR assay. The concordance of this assay with commonly used emergency use authorization (US Food and Drug Administration) manual kits and an automated high-throughput testing platform was evaluated. Further, a retrospective analysis was carried out using Viroselect on samples reported as 'inconclusive' between April and October 2020.ResultsIn total, 701 samples were tested. Concordance analysis of 477 samples demonstrated high overall agreement of Viroselect with both manual (87.6%) and automated (84.7%) assays. Also, in the retrospective analysis of 224 additional samples reported as 'inconclusive', Viroselect was able to resolve 100% (19/19) and 93.7% (192/205) of samples which had inconclusive results on manual and automated high-throughput platforms, respectively.ConclusionViroselect had high concordance with conventional assays, both manual and automated, and has potential to resolve inconclusive samples.
| S-EPMC8036167 | biostudies-literature
Project description:BackgroundThe relation between viral load and disease severity in childhood acute respiratory tract infections (ARI) is not fully understood.ObjectivesTo assess the clinical relevance of the relation between viral load, determined by cycle threshold (CT) value of real-time reverse transcription-polymerase chain reaction assays and disease severity in children with single- and multiple viral ARI.Study design582 children with ARI were prospectively followed and tested for 15 viruses. Correlations were calculated between CT values and clinical parameters.ResultsIn single viral ARI, statistically significant correlations were found between viral loads of Respiratory Syncytial Virus (RSV) and hospitalization and between viral loads of Human Coronavirus (HCoV) and a disease severity score. In multiple-viral ARI, statistically significant correlations between viral load and clinical parameters were found. In RSV-Rhinovirus (RV) multiple infections, a low viral load of RV was correlated with a high length of hospital stay and a high duration of extra oxygen use. The mean CT value for RV, HCoV and Parainfluenza virus was significantly lower in single- versus multiple infections.ConclusionAlthough correlations between CT values and clinical parameters in patients with single and multiple viral infection were found, the clinical importance of these findings is limited because individual differences in host-, viral and laboratory factors complicate the interpretation of statistically significant findings. In multiple infections, viral load cannot be used to differentiate between disease causing virus and innocent bystanders.
| S-EPMC7185604 | biostudies-literature
Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the ongoing COVID-19 pandemic, has infected millions within just a few months and is continuing to spread around the globe causing immense respiratory disease and mortality. Assays to monitor SARS-CoV-2 growth depend on time-consuming and costly RNA extraction steps, hampering progress in basic research and drug development efforts. Here we developed a facile Q-RT-PCR assay that bypasses viral RNA extraction steps and can monitor SARS-CoV-2 replication kinetics from a small amount of cell culture supernatants. Using this assay, we screened the activities of a number of entry, SARS-CoV-2- and HIV-1-specific inhibitors in a proof of concept study. In line with previous studies which has shown that processing of the viral Spike protein by cellular proteases and endosomal fusion are required for entry, we found that E64D and apilimod potently decreased the amount of SARS-CoV-2 RNA in cell culture supernatants with minimal cytotoxicity. Surprisingly, we found that macropinocytosis inhibitor EIPA similarly decreased viral RNA in supernatants suggesting that entry may additionally be mediated by an alternative pathway. HIV-1-specific inhibitors nevirapine (an NNRTI), amprenavir (a protease inhibitor), and ALLINI-2 (an allosteric integrase inhibitor) modestly inhibited SARS-CoV-2 replication, albeit the IC 50 values were much higher than that required for HIV-1. Taken together, this facile assay will undoubtedly expedite basic SARS-CoV-2 research, be amenable to mid-throughput screens to identify chemical inhibitors of SARS-CoV-2, and be applicable to a broad number of RNA and DNA viruses.
| S-EPMC7325174 | biostudies-literature
Project description:The severe acute respiratory syndrome (SARS) epidemic originating from China in 2002 was caused by a previously uncharacterized coronavirus that could be identified by specific RT-PCR amplification. Efforts to control future SARS outbreaks depend on the accurate and early identification of SARS-CoV infected patients. A real-time fluorogenic RT-PCR assay based on the 3'-noncoding region (3'-NCR) of SARS-CoV genome was developed as a quantitative SARS diagnostic tool. The ideal amplification efficiency of a sensitive SARS-CoV RT-PCR assay should yield an E value (PCR product concentration increase per amplification cycle) equal to 2.0. It was demonstrated that the 3'-NCR SARS-CoV based RT-PCR reactions could be formulated to reach excellent E values of 1.81, or 91% amplification efficacy. The SARS-CoV cDNA preparations derived from viral RNA extract and the cloned recombinant plasmid both exhibit the identical amplification characteristics, i.e. amplification efficacy using the same PCR formulation developed in this study. The viral genomic copy (or genomic equivalences, GE) per infectious unit (GE/pfu) of SARS-CoV used in this study was also established to be approximate 1200-1600:1. The assay's detection sensitivity could reach 0.005 pfu or 6-8 GE per assay. It was preliminarily demonstrated that the assay could efficiently detect SARS-CoV from clinical specimens of SARS probable and suspected patients identified in Taiwan. The 3'-NCR based SARS-CoV assay demonstrated 100% diagnostic specificity testing samples of patients with acute respiratory disease from a non-SARS epidemic region.
| S-EPMC7119649 | biostudies-literature
Project description:ObjectiveLow viral load from patients infected with SARS-CoV-2 during infection late stage easily lead to false negative nucleic acid testing results, thus having great challenges to the prevention and control of the current pandemic. In present study, we mainly aimed to evaluate specimen types and specimen collection timepoint on the positive detection of 2019 novel coronavirus from patients at infection late stage based on RT-PCR testing.MethodsPaired nasopharyngeal swabs, nasal swabs, oropharyngeal swabs and anal swabs were collected from patients infected with SARS-CoV-2 during infection late stage before washing in the morning and afternoon on the same day. Then virus RNA was extracted and tested for 2019-nCoV identification by RT-PCR within 24 h.ResultsViral load was low at late infection stage. Specimens collected before washing in the morning would increase the detection ratio of 2019-nCoV. Detection ratio of nasopharyngeal swab [65 (95 % CI: 49.51-77.87) vs 42.5(95 % CI: 28.51-57.8)] or nasal swab [57.5 (95 % CI: 42.2-71.49) vs 35 (95 % CI: 22.13-50.49)] is higher not only than oropharyngeal swab[22.5 (95 % CI: 12.32-37.5) vs 7.5 (95 % CI: 2.58-19.86)], but also anal swab[2.5 (95 % CI: 0.44-12.88) vs 5 (95 % CI: 1.38-16.5)].ConclusionsIn summary, our research discovers that nasopharyngeal or nasal swab collected before washing in the morning might be more suitable for detecting of large-scale specimens from patients infected with low SARS-CoV-2 load during infection late stage. Those results could facilitate other laboratories in collecting appropriate specimens for improving detection of SARS-CoV-2 from patients during infection late stage as well as initially screening.
| S-EPMC7493793 | biostudies-literature