scholarly journals Investigation of pooling strategies using clinical COVID-19 samples for more efficient diagnostic testing

2020 ◽  
Author(s):  
Samantha H Adikari ◽  
Emily Z Alipio Lyon ◽  
Attelia D Hollander ◽  
Alina Deshpande ◽  
Elizabeth Hong-Geller
Keyword(s):  
Diagnostic Testing ◽  
Rna Extraction ◽  
Positive Sample ◽  
Viral Rna ◽  
Clinical Samples ◽  
Extraction Column ◽  
Diagnostic Assay ◽  
Significant Difference ◽  
Pooling Strategies ◽  
Single Positive

When testing large numbers of clinical COVID-19 samples for diagnostic purposes, pooling samples together for processing can offer significant reductions in the materials, reagents, time, and labor needed. We have evaluated two different strategies for pooling independent nasopharyngeal swab samples prior to testing with an EUA-approved SARS-CoV-2 RT-qPCR diagnostic assay. First, in the Dilution Study, we assessed the assay's ability to detect a single positive clinical sample diluted in multiple negative samples before the viral RNA extraction stage. We observed that positive samples with Ct values at ~30 can be reliably detected in pools of up to 30 independent samples, and positive samples with Ct values at ~35 can be detected in pools of 5 samples. Second, in the Reloading Study, we assessed the efficacy of reloading QIAamp viral RNA extraction columns numerous times using a single positive sample and multiple negative samples. We determined that one RNA extraction column can be reloaded with up to 20 clinical samples (1 positive and 19 negatives) sequentially without any loss of signal in the diagnostic assay. Furthermore, we found there was no significant difference in assay readout whether the positive sample was loaded first or last in a series of 20 samples. These results demonstrate that different pooling strategies can lead to increased process efficiencies for COVID-19 clinical diagnostic testing.

scholarly journals Direct diagnostic testing of SARS-CoV-2 without the need for prior RNA extraction

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shan Wei ◽  
Esther Kohl ◽  
Alexandre Djandji ◽  
Stephanie Morgan ◽  
Susan Whittier ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Cost Effective ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Percentage Agreement ◽  
Testing Method ◽  
Viral Transport

AbstractThe COVID-19 pandemic has resulted in an urgent need for a rapid, point of care diagnostic testing that could be rapidly scaled on a worldwide level. We developed and tested a highly sensitive and robust assay based on reverse transcription loop mediated isothermal amplification (RT-LAMP) that uses readily available reagents and a simple heat block using contrived spike-in and actual clinical samples. RT-LAMP testing on RNA-spiked samples showed a limit of detection (LoD) of 2.5 copies/μl of viral transport media. RT-LAMP testing directly on clinical nasopharyngeal swab samples in viral transport media had an 85% positive percentage agreement (PPA) (17/20), and 100% negative percentage agreement (NPV) and delivered results in 30 min. Our optimized RT-LAMP based testing method is a scalable system that is sufficiently sensitive and robust to test for SARS-CoV-2 directly on clinical nasopharyngeal swab samples in viral transport media in 30 min at the point of care without the need for specialized or proprietary equipment or reagents. This cost-effective and efficient one-step testing method can be readily available for COVID-19 testing world-wide, especially in resource poor settings.

scholarly journals Direct Viral RNA Detection of SARS-CoV-2 and DENV in Inactivated Samples by Real-Time RT-qPCR: Implications for Diagnosis in Resource Limited Settings with Flavivirus Co-Circulation

Pathogens ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1558
Author(s):  
Zhan Qiu Mao ◽  
Mizuki Fukuta ◽  
Jean Claude Balingit ◽  
Thi Thanh Ngan Nguyen ◽  
Co Thach Nguyen ◽  
...  
Keyword(s):  
Real Time ◽  
Early Stage ◽  
Rna Extraction ◽  
Viral Rna ◽  
Clinical Samples ◽  
Heat Inactivation ◽  
Resource Limited Settings ◽  
Rna Detection ◽  
Resource Limited ◽  
Starting Point

The RT-qPCR method remains the gold standard and first-line diagnostic method for the detection of SARS-CoV-2 and flaviviruses, especially in the early stage of viral infection. Rapid and accurate viral detection is a starting point in the containment of the COVID-19 pandemic and flavivirus outbreaks. However, the shortage of diagnostic reagents and supplies, especially in resource-limited countries that experience co-circulation of SARS-CoV-2 and flaviviruses, are limitations that may result in lesser availability of RT-qPCR-based diagnostic tests. In this study, the utility of RNA-free extraction methods was assessed for the direct detection of SARS-CoV-2 and DENV-2 in heat-inactivated or chemical-inactivated samples. The findings demonstrate that direct real-time RT-qPCR is a feasible option in comparison to conventional real-time RT-qPCR based on viral genome extraction-based methods. The utility of heat-inactivation and direct real-time RT-qPCR for SARS-CoV-2, DENV-2 viral RNA detection was demonstrated by using clinical samples of SARS-CoV-2 and DENV-2 and spiked cell culture samples of SARS-CoV-2 and DENV-2. This study provides a simple alternative workflow for flavivirus and SARS-CoV-2 detection that includes heat inactivation and viral RNA extraction-free protocols, with aims to reduce the risk of exposure during processing of SARS-CoV-2 biological specimens and to overcome the supply-chain bottleneck, particularly in resource limited settings with flavivirus co-circulation.

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 Evaluation of Sample Pooling for Screening of SARS CoV-2

2020 ◽  
Author(s):  
Andargachew Mulu ◽  
Dawit Hailu Alemayehu ◽  
Fekadu Alemu ◽  
Dessalegn Abeje Tefera ◽  
Sinknesh Wolde ◽  
...  
Keyword(s):  
Ad Hoc ◽  
Clinical Sample ◽  
Diagnostic Testing ◽  
Positive Sample ◽  
Clinical Samples ◽  
Global Public Health ◽  
Rt Pcr ◽  
Resource Saving ◽  
Public Health Importance ◽  
Ct Value

AbstractBackgroundThe coronavirus disease (COVID-19) pandemic has revealed the global public health importance of robust diagnostic testing. To overcome the challenge of nucleic acid (NA) extraction and testing kit availability efficient method is urgently needed.ObjectivesTo establish an efficient, time and resource-saving and cost-effective methods, and to propose an ad hoc pooling approach for mass screening of SARS-CoV-2MethodsDirect clinical sample and NA pooling approach was used for the standard reverse transcriptase polymerase chain reaction (RT-PCR) test of the SARS CoV-2 targeting the envelop (E) and open reading frame (ORF1ab) genomic region of the virus. In this approach, experimental pools were created using SARS CoV-2 positive clinical samples spiked with up to 9 negative samples prior to NA extraction step to have a final extraction volume of 200 μL (maximum dilution factor of 10). Viral NA was also subsequently extracted from each pool and tested using the SARS CoV-2 RT-PCR assay.ResultsWe found that a single positive sample can be amplified and detected in pools of up to 7 samples depending on the ct value of the original sample, corresponding to high, medium, and low SARS CoV-2 viral copies/reaction. However, to minimize false negativity of the assay with pooling strategies and with unknown false negativity rate of the assay under validation, we recommend poling of 4 in 1 using the standard protocols of the assay, reagents and equipment. The predictive algorithm indicated a pooling ratio of 4 in 1 was expected to retain accuracy of the test irrespective of the ct value (relative RNA copy number) of the sample spiked and result in a 237% increase in testing efficiency.ConclusionsThe approaches showed its concept in easily customized and resource-saving manner and would allow expanding of current screening capacities and enable the expansion of detection in the community.

scholarly journals Reducing false-positive SARS-CoV-2 diagnoses using long-range RT-qPCR

2021 ◽  
Author(s):  
Aartjan J.W. te Velthuis ◽  
Dovile Juozapaite ◽  
Charlotte Rigby ◽  
Ingrida Olendraite ◽  
Pankaj Mathur ◽  
...  
Keyword(s):  
Rna Virus ◽  
Quantitative Polymerase Chain Reaction ◽  
Diagnostic Testing ◽  
Genetic Material ◽  
Viral Rna ◽  
Clinical Samples ◽  
Virus Infections ◽  
Molecular Method ◽  
Polymerase Chain ◽  
Positive Results

Quantitative polymerase chain reaction (qPCR) is a sensitive molecular method for the detection of genetic material and regarded as the gold-standard for diagnostic testing. To detect respiratory RNA virus infections, a reverse transcription (RT) step is implemented to create cDNA molecules that can serve as template in the qPCR step. However, positive RT-qPCR results can be found long after patient recovery, in part because the RT-qPCR can detect residual viral RNA genome fragments. To minimize the detection of such fragments, we here modified the RT-qPCR assay by replacing the routinely used random hexamers with an oligonucleotide that binds to the 3' end of the viral genome. We demonstrate that this method allows us to distinguish between infectious and non-infectious samples. Moreover, in clinical samples obtained over 15 days after the onset of symptoms, we observe that the modified RT-qPCR protocol yields significantly fewer positive results compared to a commercial RT-qPCR test. No significantly different results were found compared to the commercial test when SARS-CoV-2 clinical samples were tested within 5 days of the onset of symptoms, suggesting that the modification has a similar sensitivity for detecting infectious viral RNA. Overall, these findings may help differentiate between incorrectly-positive, persistently positive, and reinfection cases in COVID-19 patients.

scholarly journals A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of SARS-CoV-2 within nasopharyngeal and oropharyngeal swabs at Hampshire Hospitals NHS Foundation Trust

2020 ◽  
Author(s):  
Veronica L. Fowler ◽  
Bryony Armson ◽  
Jose L. Gonzales ◽  
Emma L. Wise ◽  
Emma L. A. Howson ◽  
...  
Keyword(s):  
Diagnostic Testing ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Standard Of Care ◽  
Positive Sample ◽  
Diagnostic Sensitivity ◽  
Nucleic Acid Amplification ◽  
Analytical Sensitivity ◽  
Synthetic Dna ◽  
High Prevalence

AbstractThe COVID-19 pandemic has illustrated the importance of rapid, accurate diagnostic testing for the effective triaging and cohorting of patients and timely tracking and tracing of cases. However, a surge in diagnostic testing quickly resulted in worldwide competition for the same sample preparation and real-time RT-PCR diagnostic reagents (rRT-PCR). Consequently, Hampshire Hospitals NHS Foundation Trust, UK sought to diversify their diagnostic portfolio by exploring alternative amplification chemistries including those that permit direct testing without RNA extraction. This study describes the validation of a SARS-CoV-2 RT-LAMP assay, which is an isothermal, autocycling, strand-displacement nucleic acid amplification technique which can be performed on extracted RNA, “RNA RT-LAMP” or directly from swab “Direct RT-LAMP”. Analytical specificity (ASp) of this new RT-LAMP assay was 100% and analytical sensitivity (ASe) was between 1×101 and 1×102 copies when using a synthetic DNA target. The overall diagnostic sensitivity (DSe) and specificity (DSp) of RNA RT-LAMP was 97% and 99% respectively, relative to the standard of care (SoC) rRT-PCR. When a CT cut-off of 33 was employed, above which increasingly, evidence suggests there is a very low risk of patients shedding infectious virus, the diagnostic sensitivity was 100%. The DSe and DSp of Direct-RT-LAMP was 67% and 97%, respectively. When setting CT cut-offs of ≤33 and ≤25, the DSe increased to 75% and 100%, respectively. Time from swab-to-result for a strong positive sample (CT < 25) was < 15 minutes. We propose that RNA RT-LAMP could replace rRT-PCR where there is a need for increase in throughput, whereas Direct RT-LAMP could be used as a screening tool for triaging patients into appropriate hospitals wards, at GP surgeries and in care homes, or for population screening to identify highly contagious individuals (“super shedders”). Direct RT-LAMP could also be used during times of high prevalence to save critical extraction and rRT-PCR reagents by “screening” out those strong positives from diagnostic pipelines.

scholarly journals Comparative analysis of the diagnostic performance of five commercial COVID-19 qRT PCR kits used in India

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
J. Singh ◽  
A. K. Yadav ◽  
A. Pakhare ◽  
P. Kulkarni ◽  
L. Lokhande ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Diagnostic Performance ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Clinical Samples ◽  
Diagnostic Process ◽  
Control Group ◽  
Rt Pcr ◽  
Short Period ◽  
Commercial Kits

AbstractTo meet the unprecedented requirement of diagnostic testing for SARS-CoV-2, a large number of diagnostic kits were authorized by concerned authorities for diagnostic use within a short period of time during the initial phases of the ongoing pandemic. We undertook this study to evaluate the inter-test agreement and other key operational features of 5 such commercial kits that have been extensively used in India for routine diagnostic testing for COVID-19. The five commercial kits were evaluated, using a panel of positive and negative respiratory samples, considering the kit provided by National Institute of Virology, Indian Council of Medical Research (2019-nCoV Kit) as the reference. The positive panel comprised of individuals who fulfilled the 3 criteria of being clinically symptomatic, having history of contact with diagnosed cases and testing positive in the reference kit. The negative panel included both healthy and disease controls, the latter being drawn from individuals diagnosed with other respiratory viral infections. The same protocol of sample collection, same RNA extraction kit and same RT-PCR instrument were used for all the kits. Clinical samples were collected from a panel of 92 cases and 60 control patients, who fulfilled our inclusion criteria. The control group included equal number of healthy individuals and patients infected with other respiratory viruses (n = 30, in each group). We observed varying sensitivity and specificity among the evaluated kits, with LabGun COVID-19 RT-PCR kit showing the highest sensitivity and specificity (94% and 100% respectively), followed by TaqPath COVID-19 Combo and Allplex 2019-nCoV assays. The extent of inter-test agreement was not associated with viral loads of the samples. Poor correlation was observed between Ct values of the same genes amplified using different kits. Our findings reveal the presence of wide heterogeneity and sub-optimal inter-test agreement in the diagnostic performance of the evaluated kits and hint at the need of adopting stringent standards for fulfilling the quality assurance requirements of the COVID-19 diagnostic process.

scholarly journals Evaluation of sample pooling for screening of SARS CoV-2

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0247767
Author(s):  
Andargachew Mulu ◽  
Dawit Hailu Alemayehu ◽  
Fekadu Alemu ◽  
Dessalegn Abeje Tefera ◽  
Sinknesh Wolde ◽  
...  
Keyword(s):  
Clinical Sample ◽  
Diagnostic Testing ◽  
Positive Sample ◽  
Clinical Samples ◽  
Global Public Health ◽  
Rt Pcr ◽  
Resource Saving ◽  
Public Health Importance ◽  
Ct Value ◽  
Sample Pooling

Background The coronavirus disease 2019 (COVID-19) pandemic has revealed the global public health importance of robust diagnostic testing. To overcome the challenge of nucleic acid (NA) extraction and testing kit availability, an efficient method is urgently needed. Objectives To establish an efficient, time and resource-saving and cost-effective methods, and to propose an ad hoc pooling approach for mass screening of SARS-CoV-2. Methods We evaluated pooling approach on both direct clinical and NA samples. The standard reverse transcriptase polymerase chain reaction (RT-PCR) test of the SARS CoV-2 was employed targeting the nucleocapsid (N) and open reading frame (ORF1ab) genomic region of the virus. The experimental pools were created using SARS CoV-2 positive clinical samples and extracted RNA spiked with up to 9 negative samples. For the direct clinical samples viral NA was extracted from each pool to a final extraction volume of 200μL, and subsequently both samples tested using the SARS CoV-2 RT-PCR assay. Results We found that a single positive sample can be amplified and detected in pools of up to 7 samples depending on the cycle threshold (Ct) value of the original sample, corresponding to high, and low SARS CoV-2 viral copies per reaction. However, to minimize false negativity of the assay with pooling strategies and with unknown false negativity rate of the assay under validation, we recommend pooling of 4/5 in 1 using the standard protocols of the assay, reagents and equipment. The predictive algorithm indicated a pooling ratio of 5 in 1 was expected to retain accuracy of the test irrespective of the Ct value samples spiked, and result in a 137% increase in testing efficiency. Conclusions The approaches showed its concept in easily customized and resource-saving manner and would allow expanding of current screening capacities and enable the expansion of detection in the community. We recommend clinical sample pooling of 4 or 5 in 1. However, we don’t advise pooling of clinical samples when disease prevalence is greater than 7%; particularly when sample size is large.

Environmental Swabs as a Tool in Norovirus Outbreak Investigation, Including Outbreaks on Cruise Ships

2009 ◽  
Vol 72 (1) ◽  
pp. 111-119 ◽  
Author(s):  
INGEBORG L. A. BOXMAN ◽  
REMCO DIJKMAN ◽  
NATHALIE A. J. M. te LOEKE ◽  
GEKE HÄGELE ◽  
JEROEN J. H. C. TILBURG ◽  
...  
Keyword(s):  
Detection Rate ◽  
Clinical Symptoms ◽  
Rna Extraction ◽  
Viral Rna ◽  
Food Preparation ◽  
Clinical Samples ◽  
Viral Gastroenteritis ◽  
Clinical Specimens ◽  
Cruise Ships ◽  
Set Up

In this study, we investigated whether environmental swabs can be used to demonstrate the presence of norovirus in outbreak settings. First, a procedure was set up based on viral RNA extraction using guanidium isothiocyanate buffer and binding of nucleic acids to silica. Subsequently, environmental swabs were taken at 23 Dutch restaurants and four cruise ships involved in outbreaks of gastroenteritis. Outbreaks were selected based on clinical symptoms consistent with viral gastroenteritis and time between consumption of suspected food and onset of clinical symptoms (&gt;12 h). Norovirus RNA was demonstrated by real-time reverse transcriptase PCR in 51 of 86 (59%) clinical specimens from 12 of 14 outbreaks (86%), in 13 of 90 (14%) food specimens from 4 of 18 outbreaks (22%), and in 48 of 119 (40%) swab specimens taken from 14 of 27 outbreaks (52%). Positive swab samples agreed with positive clinical samples in seven outbreaks, showing identical sequences. Furthermore, norovirus was detected on swabs taken from kitchen and bathroom surfaces in five outbreaks in which no clinical samples were collected and two outbreaks with negative fecal samples. The detection rate was highest for outbreaks associated with catered meals and lowest for restaurant-associated outbreaks. The use of environmental swabs may be a useful tool in addition to testing of food and clinical specimens, particularly when viral RNA is detected on surfaces used for food preparation.

scholarly journals Field-deployable, rapid diagnostic testing of saliva for SARS-CoV-2

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shan Wei ◽  
Hemant Suryawanshi ◽  
Alexandre Djandji ◽  
Esther Kohl ◽  
Stephanie Morgan ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Diagnostic Testing ◽  
Clinical Samples ◽  
Clinical Validation ◽  
High Complexity ◽  
Rt Pcr ◽  
Testing Methods ◽  
Rapid Diagnostic Testing ◽  
Single Positive ◽  
Fluid Transfer

AbstractTo safely re-open economies and prevent future outbreaks, rapid, frequent, point-of-need, SARS-CoV-2 diagnostic testing is necessary. However, existing field-deployable COVID-19 testing methods require the use of uncomfortable swabs and trained providers in PPE, while saliva-based methods must be transported to high complexity laboratories for testing. Here, we report the development and clinical validation of High-Performance Loop-mediated isothermal Amplification (HP-LAMP), a rapid, saliva-based, SARS-CoV-2 test with a limit of detection of 1.4 copies of virus per µl of saliva and a sensitivity and specificity with clinical samples of > 96%, on par with traditional RT-PCR based methods using swabs, but can deliver results using only a single fluid transfer step and simple heat block. Testing of 120 patient samples in 40 pools comprised of 5 patient samples each with either all negative or a single positive patient sample was 100% accurate. Thus, HP-LAMP may enable rapid and accurate results in the field using saliva, without need of a high-complexity laboratory.

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