Limited clinical value of early repeat RT-PCR testing for SARS-CoV-2.
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ABSTRACT:
SUBMITTER: Williams E
PROVIDER: S-EPMC8242732 | biostudies-literature |
REPOSITORIES: biostudies-literature
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| S-EPMC7845522 | biostudies-literature
Project description:Alternatives to nasopharyngeal sampling are needed to increase capacity for SARS-CoV-2 testing. Among 275 participants, we piloted the collection of nasal mid-turbinate swabs amenable to self-testing, including polyester flocked swabs as well as 3D-printed plastic lattice swabs, placed into viral transport media or an RNA stabilization agent. Flocked nasal swabs identified 104/121 individuals who were PCR-positive for SARS-CoV-2 by nasopharyngeal sampling (sensitivity 87%, 95% CI 79-92%), missing those with low viral load (<106 viral copies/mL). 3D-printed nasal swabs showed similar sensitivity. When nasal swabs were placed directly into RNA preservative, the mean 1.4 log decrease in viral copies/uL compared to nasopharyngeal samples was reduced to <1 log, even when samples were left at room temperature for up to 7 days. We also evaluated pooling strategies that involved pooling specimens in the lab versus pooling swabs at the point of collection, finding both successfully detected samples with >105 viral copies/mL.
| S-EPMC8230941 | biostudies-literature
Project description:Standard nasopharyngeal swab testing for SARS-CoV-2 detection by PCR is not always feasible due to limitations in trained personnel, personal protective equipment, swabs, PCR reagents, and access to cold chain and biosafety hoods. We piloted the collection of nasal mid-turbinate swabs amenable to self-testing, including both standard polyester flocked swabs as well as 3D printed plastic lattice swabs, placed into either viral transport media or an RNA stabilization agent. Quantitative SARS-CoV-2 viral detection by RT-qPCR was compared to that obtained by nasopharyngeal sampling as the reference standard. Pooling specimens in the lab versus pooling swabs at the point of collection was also evaluated. Among 275 participants, flocked nasal swabs identified 104/121 individuals who were PCR-positive for SARS-CoV-2 by nasopharyngeal sampling (sensitivity 87%, 95% CI 79-92%), mostly missing those with low viral load (<10^3 viral copies/uL). 3D-printed nasal swabs showed similar sensitivity. When nasal swabs were placed directly into an RNA stabilizer, the mean 1.4 log decrease in viral copies/uL compared to nasopharyngeal samples was reduced to <1 log, even when samples were left at room temperature for up to 7 days. Pooling sample specimens or swabs both successfully detected samples >102 viral copies/uL. Nasal swabs are likely adequate for clinical diagnosis of acute infections to help expand testing capacity in resource-constrained settings. When collected into an RNA preservative that also inactivates infectious virus, nasal swabs yielded quantitative viral loads approximating those obtained by nasopharyngeal sampling.
| S-EPMC7924294 | biostudies-literature
Project description:Severe acute respiratory syndrome (SARS) is an acute newly emerged infectious respiratory illness. The etiologic agent of SARS was named 'SARS-associated coronavirus' (SARS-CoV) that can be detected with reverse transcription-polymerase chain reaction (RT-PCR) assays. In this study, 12 sets of nested primers covering the SARS-CoV genome have been screened and showed sufficient sensitivity to detect SARS-CoV in RNA isolated from virus cultured in Vero 6 cells. To optimize further the reaction condition of those nested primers sets, seven sets of nested primers have been chosen to compare their reverse transcribed efficiency with specific and random primers, which is useful to combine RT with the first round of PCR into a one-step RT-PCR. Based on the sensitivity and simplicity of results, the no. 73 primer set was chosen as the candidate primer set for clinical diagnoses. To specify the amplicon to minimize false positive results, a Taqman RT-nested PCR system of no. 73 nested primer set was developed. Through investigations on a test panel of whole blood obtained from 30 SARS patients and 9 control persons, the specificity and sensitivity of the Taqman RT-nested PCR system was found to be 100 and 83%, respectively, which suggests that the method is a promising one to diagnose SARS in early stages.
| S-EPMC7119531 | 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
Project description:RT-PCR tests based on RNA extraction from nasopharyngeal swabs (NPS) are promoted as the "gold standard" for SARS-CoV-2 detection. However, the use of saliva samples offers noninvasive self-collection more suitable for high-throughput testing. This study evaluated performance of the TaqPath COVID-19 Fast PCR Combo kit 2.0 assay for detection of SARS-CoV-2 in raw saliva relative to a lab-developed direct RT-PCR test (SalivaDirect-based PCR, SDB-PCR) and an RT-PCR test based on RNA extraction from NPS. Saliva and NPS samples were collected from symptomatic and asymptomatic individuals (N = 615). Saliva samples were tested for SARS-CoV-2 using the TaqPath COVID-19 Fast PCR Combo kit 2.0 and the SDB-PCR, while NPS samples were tested by RT-PCR in RNA extracts according to the Irish national testing system. TaqPath COVID-19 Fast PCR Combo kit 2.0 detected SARS-CoV-2 in 52 saliva samples, of which 51 were also positive with the SDB-PCR. Compared to the NPS "gold standard" biospecimen method, 49 samples displayed concordant results, while three samples (35<Ct<37) were positive on raw saliva. Among the negative samples, 10 discordant cases were found with the TaqPath COVID-19 Fast PCR Combo kit 2.0 (PPA-83.05%; NPA-99.44%), compared to the RNA extraction-based NPS method, performing similarly to the SDB-PCR (PPA-84.75%; NPA-99.63%). The direct RT-PCR testing of saliva samples shows high concordance with the NPS extraction-based method for SARS-CoV-2 detection, and therefore provides a cost-effective and highly scalable system for high-throughput COVID-19 rapid-testing. IMPORTANCE The scale of the COVID-19 pandemic highlighted the need for viral diagnostic systems that are accurate and could be deployed at large population scales. Large-scale diagnostic or surveillance testing of large numbers of people requires collection of infected biological samples that is easy and rapid. Here, we demonstrate that raw saliva samples can be easily collected and tested by RT-PCR assays. Indeed, we find that direct testing of raw saliva by two different RT-PCR assays is as accurate (if not more accurate) than nasal swab-based RT-PCR testing. We present a cost-effective and highly scalable system for high-throughput COVID-19 rapid-testing.
| S-EPMC9769602 | biostudies-literature
Project description:Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is commonly diagnosed by reverse transcription polymerase chain reaction (RT-PCR) to detect viral RNA in patient samples, but RNA extraction constitutes a major bottleneck in current testing. Methodological simplification could increase diagnostic availability and efficiency, benefitting patient care and infection control. Here, we describe methods circumventing RNA extraction in COVID-19 testing by performing RT-PCR directly on heat-inactivated or lysed samples. Our data, including benchmarking using 597 clinical patient samples and a standardised diagnostic system, demonstrate that direct RT-PCR is viable option to extraction-based tests. Using controlled amounts of active SARS-CoV-2, we confirm effectiveness of heat inactivation by plaque assay and evaluate various generic buffers as transport medium for direct RT-PCR. Significant savings in time and cost are achieved through RNA-extraction-free protocols that are directly compatible with established PCR-based testing pipelines. This could aid expansion of COVID-19 testing.
| S-EPMC7511968 | biostudies-literature
Project description:Early diagnosis of SARS-CoV-2 is fundamental to reduce the risk of community transmission and mortality, as well as public sector expenditures. Three years after the onset of the SARS-CoV-2 pandemic, there are still gaps on what is known regarding costs and cost drivers for the major diagnostic testing strategies in low- middle-income countries (LMICs). This study aimed to estimate the cost of SARS-CoV-2 diagnosis of symptomatic suspected patients by reverse transcription polymerase chain reaction (RT-PCR) and antigen rapid diagnostic tests (Ag-RDT) in Mozambique. We conducted a retrospective cost analysis from the provider's perspective using a bottom-up, micro-costing approach, and compared the direct costs of two nasopharyngeal Ag-RDTs (Panbio and Standard Q) against the costs of three nasal Ag-RDTs (Panbio, COVIOS and LumiraDx), and RT-PCR. The study was undertaken from November 2020 to December 2021 in the country's capital city Maputo, in four healthcare facilities at primary, secondary and tertiary levels of care, and at one reference laboratory. All the resources necessary for RT-PCR and Ag-RDT tests were identified, quantified, valued, and the unit costs per test and per facility were estimated. Our findings show that the mean unit cost of SARS-CoV-2 diagnosis by nasopharyngeal Ag-RDTs was MZN 728.00 (USD 11.90, at 2020 exchange rates) for Panbio and MZN 728.00 (USD 11.90) for Standard Q. For diagnosis by nasal Ag-RDTs, Panbio was MZN 547.00 (USD 8.90), COVIOS was MZN 768.00 (USD 12.50), and LumiraDx was MZN 798.00 (USD 13.00). Medical supplies expenditures represented the main driver of the final cost (>50%), followed by personnel and overhead costs (mean 15% for each). The mean unit cost regardless of the type of Ag-RDT was MZN 714.00 (USD 11.60). Diagnosis by RT-PCR cost MZN 2,414 (USD 39.00) per test. Our sensitivity analysis suggests that focussing on reducing medical supplies costs would be the most cost-saving strategy for governments in LMICs, particularly as international prices decrease. The cost of SARS-CoV-2 diagnosis using Ag-RDTs was three times lower than RT-PCR testing. Governments in LMICs can include cost-efficient Ag-RDTs in their screening strategies, or RT-PCR if international costs of such supplies decrease further in the future. Additional analyses are recommended as the costs of testing can be influenced by the sample referral system.
| S-EPMC10263322 | biostudies-literature
Project description:Introductionthe emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of acute respiratory disease (COVID-19). SARS-CoV-2 is a positive-strand RNA virus and its genomic characterization has played a vital role in the design of appropriate diagnostics tests. The current RT-PCR protocol for SARS-CoV-2 detects two regions of the viral genome, requiring RNA extraction and several hours. There is a need for fast, simple, and cost-effective detection strategies.Methodswe optimized a protocol for direct RT-PCR detection of SARS-CoV-2 without the need for nucleic acid extraction. Nasopharyngeal samples were diluted to 1:3 using diethyl pyrocarbonate (DEPC)-treated water. The diluted samples were incubated at 95 °C for 5 min in a thermal cycler, followed by a cooling step at 4 °C for 5 min. Samples then underwent reverse transcription real-time RT-PCR in the E and RdRp genes.Resultsour direct detection protocol showed 100% concordance with the standard protocol with an average Ct value difference of 4.38 for the E region and 3.85 for the RdRp region.Conclusionthe direct PCR technique was found to be a reliable and sensitive method that can be used to reduce the time and cost of the assay by removing the need for RNA extraction. It enables the use of the assay in research, diagnostics, and screening for COVID-19 in regions with fewer economic resources, where supplies are more limited allowing for wider use for screening.
| S-EPMC7823392 | biostudies-literature
Project description:BackgroundUnderstanding the false negative rates of SARS-CoV-2 RT-PCR testing is pivotal for the management of the COVID-19 pandemic and it has implications for patient management. Our aim was to determine the real-life clinical sensitivity of SARS-CoV-2 RT-PCR.MethodsThis population-based retrospective study was conducted in March-April 2020 in the Helsinki Capital Region, Finland. Adults who were clinically suspected of SARS-CoV-2 infection and underwent SARS-CoV-2 RT-PCR testing, with sufficient data in their medical records for grading of clinical suspicion were eligible. In addition to examining the first RT-PCR test of repeat-tested individuals, we also used high clinical suspicion for COVID-19 as the reference standard for calculating the sensitivity of SARS-CoV-2 RT-PCR.ResultsAll 1,194 inpatients (mean [SD] age, 63.2 [18.3] years; 45.2% women) admitted to COVID-19 cohort wards during the study period were included. The outpatient cohort of 1,814 individuals (mean [SD] age, 45.4 [17.2] years; 69.1% women) was sampled from epidemiological line lists by systematic quasi-random sampling. The sensitivity (95% CI) for laboratory confirmed cases (repeat-tested patients) was 85.7% (81.5-89.1%) inpatients; 95.5% (92.2-97.5%) outpatients, 89.9% (88.2-92.1%) all. When also patients that were graded as high suspicion but never tested positive were included in the denominator, the sensitivity (95% CI) was: 67.5% (62.9-71.9%) inpatients; 34.9% (31.4-38.5%) outpatients; 47.3% (44.4-50.3%) all.ConclusionsThe clinical sensitivity of SARS-CoV-2 RT-PCR testing was only moderate at best. The relatively high false negative rates of SARS-CoV-2 RT-PCR testing need to be accounted for in clinical decision making, epidemiological interpretations, and when using RT-PCR as a reference for other tests.
| S-EPMC8139477 | biostudies-literature