Pooling Nasopharyngeal Swab Specimens to Improve Testing Capacity for SARS-CoV-2 by Real-Time RT-PCR

Abstract Background The detection of SARS-CoV-2 using qRT-PCR with the pooling of samples can reduce workload and costs especially when the prevalence rate of COVID-19 in a population is low. To analyse the effect of pooling samples on the sensitivity of RT-qPCR for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection, we compared the cycle threshold (Ct) values of pools of 5 and 10 that tested positive with Ct values of individual samples that tested positive in that pool. Twenty positive nasopharyngeal (NP) specimens with low to high viral load were selected and pooled individually with four and nine negative NP. Results In NP specimens, the sensitivity of pools of 5 and 10 were 90 and 85%, compared to individual sample testing, respectively. The RT-qPCR sensitivity of pools of 5 and 10 against individual testing were not significantly different (p > 0.05). Detection of positive samples with low Ct values (< 36) was consistently achieved in pools of 5 and 10. However, there were higher false negatives when samples with high ct values (> 36) were pooled and tested. The mean Ct values obtained with the 5-sample pooled testing exceeded individual sample testing by 1.85 ± 1.09 cycles, while Ct values obtained with the 10-sample pooling exceeded individual sample testing by 3.4 ± 1.65 cycles. Conclusions In a low prevalence setting, testing capacity can be increased by pooling 5 or 10 samples, but the risk of additional false negatives needs to be considered

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Effectiveness of Sample Pooling Strategies for SARS-CoV-2 Mass Screening by RT-PCR: A Scoping Review

Journal of Laboratory Physicians ◽

10.1055/s-0040-1721159 ◽

2020 ◽

Vol 12 (03) ◽

pp. 212-218 ◽

Cited By ~ 1

Author(s):

Sangeeta Deka ◽

Deepjyoti Kalita

Keyword(s):

Limit Of Detection ◽

Large Population ◽

Pool Size ◽

High Threshold ◽

Conservation Of Resources ◽

Rt Pcr ◽

Sample Pooling ◽

Sample Testing ◽

Low Prevalence ◽

Pooled Sample

AbstractThe ongoing COVID-19 pandemic has hugely impacted the economy of many countries, and there is an acute shortage of diagnostic resources. With the exponential increase in the number of cases and necessity to screen large number of people, there is a steep increase in the demand for diagnostic kits. Pooled-sample testing is a promising strategy to screen a large population rapidly with limited resources. The aim of this work was to compile a cohesive literature review of the effectiveness and accuracy of pooled-sample testing in the detection of SARS-CoV-2 and critically analyze its limitations. Medline, Google Scholar, Embase, and preprint servers (e.g., bioRxiv) were searched for literature on pooled testing for diagnosis of COVID-19, and out of initial 60 articles/reports, nine original articles were retained. Optimal pool size (number of samples in a pool) seemed to be dependent on factors like prevalence or rate of positivity in community. In low-prevalence localities pool size of around 30 seemed to be effective as observed by some authors. All the researchers had found significant reduction in number of tests (depending on pool size, stages, and pooling design), leading to conservation of resources. Pooling can be done with extracted RNA eluate or directly with patient’s sample before extraction. This leads to further reduction in consumables, time and manpower. Risk of false negativity in samples with high-threshold cycle (i.e., low-viral load) value was a concern. Some researchers suggest adding few additional cycles to lower the chances of missing positive cases with low-Ct value. Lower limit of detection (LoD) of RT-PCR kits, that is, sensitivity of kits was another factor to consider. Thus, in a country like India, given the economic benefit and scarcity of resources, pooling strategy can be very effective, especially in low-prevalence areas and in low-risk contacts.

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Pooled Sample Testing for SARS-CoV-2 Using Rapid RT-PCR COVID-19 Tests

10.1101/2021.01.22.21250339 ◽

2021 ◽

Author(s):

Bethany Hyde ◽

Prat Verma ◽

Ethan M. Berke

Keyword(s):

Lower Cost ◽

Limit Of Detection ◽

Point Of Care ◽

Rapid Testing ◽

Rt Pcr ◽

Pooled Testing ◽

Testing Performance ◽

Sample Testing ◽

Pooled Sample ◽

Pool Sizes

AbstractWe tested an operationally efficient way to pool samples on a rapid, point-of-care PCR device and examined the limit of detection of SARS-CoV-2 for various pool sizes. Pooled testing maintained testing performance similar to individual sample PCR testing, offering the potential for scalable rapid testing at lower cost with less supplies.

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Pooling of Nasopharyngeal Swab Specimens for SARS-CoV-2 detection by RT-PCR

10.1101/2020.04.22.20075598 ◽

2020 ◽

Cited By ~ 5

Author(s):

Ignacio Torres ◽

Eliseo Albert ◽

David Navarro

Keyword(s):

Large Population ◽

Rdrp Gene ◽

Contact Tracing ◽

Nasopharyngeal Swab ◽

Rt Pcr ◽

Case Identification ◽

E Gene ◽

Sample Pooling ◽

Community Transmission ◽

Pcr Targeting

ABSTRACTSystematic testing of large population groups by RT-PCR is mandatory to Covid-19 case identification and contact tracing in order to minimize the likelyhood of resurgence in contagion. Sample pooling for RT-PCR has been effectively used to detect community transmission of SARS CoV-2. Nevertheless, this procedure may decrease the sensitivity of RT-PCR assays due to specimen dilution. We evaluated the efficacy of this strategy for diagnosis of Covid-19 using a sensitive commercially-available RT-PCR targeting SARS CoV-2 E and RdRp genes in a single reaction. A total of 20 mini-pools containing either 5 (n=10) or 10 (n=10) nasopharyngeal exudates collected in universal transport medium were made, each of which including a unique positive NP specimen. Positive specimens yielding CT <32 for the E gene (6 out of 10) or <35.2 for the RdRP gene (7 out of 10) were detected in mini-pools of both sizes. In contrast, most NP samples displaying CTs > 35.8 for the E gene or 35.7 for the RdRP gene remained undetected in mini-pools of 5 specimens (3/4 and 2/3, respectively) or in mini-pools of 10 samples (4/4 and 3/3, respectively.

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Is the COVID-19 disease associated with de novo nephritic syndrome?

Revista da Associação Médica Brasileira ◽

10.1590/1806-9282.66.9.1258 ◽

2020 ◽

Vol 66 (9) ◽

pp. 1258-1263

Author(s):

Hamad Dheir ◽

Savas Sipahi ◽

Selcuk Yaylaci ◽

Ahmed Cihad Genc ◽

Fevziye Turkoglu Genc ◽

...

Keyword(s):

De Novo ◽

Spontaneous Remission ◽

Cross Sectional Study ◽

Nasopharyngeal Swab ◽

Rt Pcr ◽

Dipstick Test ◽

Cross Sectional ◽

Nephritic Syndrome ◽

The Mean

SUMMARY INTRODUCTION: This study aims to determine the incidence of de novo nephritic syndrome (NS) in COVID-19 patients and identify its associated factors. METHODS: All ward patients with COVID-19 pneumonia were investigated. After determining the inclusion and exclusion criteria, the study population was identified. The urine dipstick test and urine protein creatinine ratio (UPCR) measurements were performed. Patients with de novo NS findings, nasopharyngeal swab, and urine RT-PCR tests were performed simultaneously RESULTS: This descriptive cross-sectional study was conducted with 21 patients with COVID-19. The mean age of the patients was 42.2±8.8 years, and 71.4% of them were male. The mean duration of follow-up was 28.4±9.3 days. The urine RT-PCR test was positive in one patient (4.8%). Improvements were observed in hematuria by 71.4%, and proteinuria by 85.7% at the end of the follow-up. A significant decrease in the measured UPCR was found in comparison to the baseline(P=0.000). Also, improvements were recorded in the complete blood counts, inflammatory parameters, ferritin, and coagulation tests, compared to the baseline. There was a positive correlation between baseline UPCR and ferritin, and a negative correlation between baseline UPCR and sodium values CONCLUSION: COVID-19-induced de novo nephritic syndrome may occur mainly due to tubulointerstitial involvement and often results in spontaneous remission. However, why these findings were not present in all patients who had no comorbidities is not clear.

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Head-to-head comparison of SARS-CoV-2 antigen-detecting rapid test with self-collected anterior nasal swab versus professional-collected nasopharyngeal swab

10.1101/2020.10.26.20219600 ◽

2020 ◽

Cited By ~ 11

Author(s):

Andreas K. Lindner ◽

Olga Nikolai ◽

Franka Kausch ◽

Mia Wintel ◽

Franziska Hommes ◽

...

Keyword(s):

Point Of Care ◽

Rapid Test ◽

High Viral Load ◽

Ease Of Use ◽

Healthcare Personnel ◽

Nasopharyngeal Swab ◽

Swab Sample ◽

Rt Pcr ◽

Percent Agreement ◽

Accuracy Study

AbstractBackgroundTwo antigen-detecting rapid diagnostic tests (Ag-RDTs) are now approved through the WHO Emergency Use Listing procedure and can be performed at the point-of-care. However, both tests use nasopharyngeal (NP) swab samples. NP swab samples must be collected by trained healthcare personnel with protective equipment and are frequently perceived as uncomfortable by patients.MethodsThis was a manufacturer-independent, prospective diagnostic accuracy study with comparison of a supervised, self-collected anterior nose (AN) swab sample with a professional-collected NP swab sample, using a WHO-listed SARS-CoV-2 Ag-RDT, STANDARD Q COVID-19 Ag Test (SD Biosensor), which is also being distributed by Roche. The reference standard was RT-PCR from an oro-/nasopharyngeal swab sample. Percent positive and negative agreement as well as sensitivity and specificity were calculated.ResultsAmong the 289 participants, 39 (13.5%) tested positive for SARS-CoV-2 by RT-PCR. The positive percent agreement of the two different sampling techniques for the Ag-RDT was 90.6% (CI 75.8-96.8). The negative percent agreement was 99.2% (CI 97.2-99.8). The Ag-RDT with AN sampling showed a sensitivity of 74.4% (29/39 PCR positives detected; CI 58.9-85.4) and specificity of 99.2% (CI 97.1-99.8) compared to RT-PCR. The sensitivity with NP sampling was 79.5% (31/39 PCR positives detected; CI 64.5-89.2) and specificity was 99.6% (CI 97.8-100). In patients with high viral load (>7.0 log 10 RNA SARS-CoV2/swab), the sensitivity of the Ag-RDT with AN sampling was 96% and 100% with NP sampling.ConclusionSupervised self-sampling from the anterior nose is a reliable alternative to professional nasopharyngeal sampling using a WHO-listed SARS-CoV-2 Ag-RDT. Considering the ease-of-use of Ag-RDTs, self-sampling and potentially patient self-testing at home may be a future use case.

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Immunological assessment of SARS-CoV-2 Infection in Pregnancy From Diagnosis to Delivery: A multicentre prospective observational study

10.22541/au.160554794.41545816/v1 ◽

2020 ◽

Author(s):

Kate Glennon ◽

Jennifer Donnelly ◽

Susan Knowles ◽

Fionnuala McAuliffe ◽

Alma O Reilly ◽

...

Keyword(s):

Antibody Response ◽

Prospective Observational Study ◽

Passive Immunity ◽

Nasopharyngeal Swab ◽

Rt Pcr ◽

Low Prevalence ◽

Fetal Response ◽

Study Setting ◽

Multicentre Prospective Study ◽

In Pregnancy

Objective We profile the maternal and fetal response to SARS-CoV-2 infection in symptomatic and asymptomatic pregnant women and make an assessment of passive immunity to the neonate, Design Multicentre prospective study. Setting Dublin, Ireland Methods RT-PCR for viral RNA via a nasopharyngeal swab was performed using the Cobas SARS-CoV-2 6800 platform. Maternal, and fetal serological antibody response, via umbilical cord bloods, was measured using both the Elecsys® immunoassay, Abbott SARS-CoV-2 IgG Assay and the IgM Architect assay. Main outcome Measure Prevalence of RT PCR positive SARS-CoV-2. Assessment of IgM and IgG anti-SARS-CoV-2 serology antibodies. Results Ten of twenty three symptomatic women had SARS-CoV-2 RNA in a nasopharyngeal swab. Five (5/23, 21.7%) demonstrated serological evidence of anti-SARS-CoV-2 IgG antibodies and seven (30.4%, 7/23) were positive for IgM antibodies. In the asymptomatic cohort, the prevalence of SARS-CoV-2 infection in RNA was 0.16% (1/608). IgG SARS-CoV-2 antibodies were detected in 1·67% (10/598, 95% CI 0·8%-3·1%) and IgM in 3·51% (21/598, 95% CI 2·3–5·5%). Nine women had repeat testing between post baseline. Four (4/9, 44%) remained IgM positive, one IgG positive. IgG anti SARS-CoV-2 antibodies were detectable in cord bloods from babies born to five seropositive women who delivered during the study. Conclusion Using two independent serological assays, we present a comprehensive illustration of the antibody response to SARS-CoV-2 in pregnancy, and show a low prevalence of asymptomatic SARS-CoV-2. Transplacental migration of anti-SARS-CoV-2 antibodies was identified in cord blood of women who demonstrated antenatal anti-SARS-CoV-2 antibodies, raising the possibility of passive immunity.

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Head-to-head performance comparison of self-collected nasal versus professional-collected nasopharyngeal swab for a WHO-listed SARS-CoV-2 antigen-detecting rapid diagnostic test

Medical Microbiology and Immunology ◽

10.1007/s00430-021-00710-9 ◽

2021 ◽

Author(s):

Julian A. F. Klein ◽

◽

Lisa J. Krüger ◽

Frank Tobian ◽

Mary Gaeddert ◽

...

Keyword(s):

High Viral Load ◽

Performance Comparison ◽

World Health ◽

Nasopharyngeal Swab ◽

Rapid Diagnostics ◽

Sample Collection ◽

Rt Pcr ◽

Percent Agreement ◽

Polymerase Chain ◽

Health Organization

AbstractIn 2020, the World Health Organization (WHO) recommended two SARS-CoV-2 lateral flow antigen-detecting rapid diagnostics tests (Ag-RDTs), both initially with nasopharyngeal (NP) sample collection. Independent head-to-head studies are necessary for SARS-CoV-2 Ag-RDT nasal sampling to demonstrate comparability of performance with nasopharyngeal (NP) sampling. We conducted a head-to-head comparison study of a supervised, self-collected nasal mid-turbinate (NMT) swab and a professional-collected NP swab, using the Panbio™ Ag-RDT (distributed by Abbott). We calculated positive and negative percent agreement between the sampling methods as well as sensitivity and specificity for both sampling techniques compared to the reference standard reverse transcription polymerase chain reaction (RT-PCR). A SARS-CoV-2 infection could be diagnosed by RT-PCR in 45 of 290 participants (15.5%). Comparing the NMT and NP sampling the positive percent agreement of the Ag-RDT was 88.1% (37/42 PCR positives detected; CI 75.0–94.8%). The negative percent agreement was 98.8% (245/248; CI 96.5–99.6%). The overall sensitivity of Panbio with NMT sampling was 84.4% (38/45; CI 71.2–92.3%) and 88.9% (40/45; CI 76.5–95.5%) with NP sampling. Specificity was 99.2% (243/245; CI 97.1–99.8%) for both, NP and NMT sampling. The sensitivity of the Panbio test in participants with high viral load (> 7 log10 SARS-CoV-2 RNA copies/mL) was 96.3% (CI 81.7–99.8%) for both, NMT and NP sampling. For the Panbio supervised NMT self-sampling yields comparable results to NP sampling. This suggests that nasal self-sampling could be used for to enable scaled-up population testing.Clinical Trial DRKS00021220.

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Head-to-head comparison of SARS-CoV-2 antigen-detecting rapid test with professional-collected anterior nasal versus nasopharyngeal swab

10.1101/2020.12.03.20243725 ◽

2020 ◽

Author(s):

Andreas K. Lindner ◽

Olga Nikolai ◽

Chiara Rohardt ◽

Susen Burock ◽

Claudia Hülso ◽

...

Keyword(s):

Healthcare Professionals ◽

Rapid Test ◽

High Viral Load ◽

Nasopharyngeal Swab ◽

Rt Pcr ◽

Test Procedures ◽

Testing Strategies ◽

Percent Agreement ◽

Accuracy Study ◽

Antigen Testing

AbstractBackgroundNasopharyngeal (NP) swab samples for antigen-detecting rapid diagnostic tests (Ag-RDTs) require qualified healthcare professionals and are frequently perceived as uncomfortable by patients.MethodsWe performed a manufacturer-independent, prospective diagnostic accuracy study, comparing professional-collected anterior nasal (AN) to nasopharyngeal swab, using the test kits of a WHO-listed SARS-CoV-2 Ag-RDT (STANDARD Q COVID-19 Ag Test, SD Biosensor), which is also being distributed by Roche. Individuals with high suspicion for COVID-19 infection were tested. The reference standard was RT-PCR using a combined oro-/nasopharyngeal swab sample. Percent positive and negative agreement, as well as sensitivity and specificity were calculated.ResultsAmong the 179 participants, 41 (22.9%) tested positive for SARS-CoV-2 by RT-PCR. The positive percent agreement of the two different sampling techniques for the Ag-RDT was 93.5% (CI 79.3-98.2). The negative percent agreement was 95.9% (CI 91.4-98.1). The Ag-RDT with AN-sampling showed a sensitivity of 80.5% (33/41 PCR positives detected; CI 66.0-89.8) and specificity of 98.6% (CI 94.9-99.6) compared to RT-PCR. The sensitivity with NP-sampling was 73.2% (30/41 PCR positives detected; CI 58.1-84.3) and specificity was 99.3% (CI 96.0-100). In patients with high viral load (>7.0 log10 RNA SARS-CoV2/swab), the sensitivity of the Ag-RDT with AN-sampling was 100% and 94.7% with NP-sampling.ConclusionThis study demonstrates that sensitivity of a WHO-listed SARS-CoV-2 Ag-RDT using a professional AN-sampling kit is at least equal to that of the NP-sampling kit, although confidence intervals overlap. Of note, differences in the IFUs of the test procedures could have contributed to different sensitivities. AN-sampling can be performed with less training, reduces patient discomfort, and it enables scaling of antigen testing strategies. Additional studies of patient self-sampling should be considered to further facilitate the scaling-up of Ag-RDT testing.

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Metagenome of SARS-Cov2 from a patient in Brazil shows a wide range of bacterial species - Lautropia, Prevotella, Haemophilus - overshadowing viral reads, which does not even add up to a full genome, explaining false negatives

10.31219/osf.io/2xt3w ◽

2020 ◽

Author(s):

Sandeep Chakraborty

Keyword(s):

Bacterial Species ◽

Bacterial Load ◽

San Diego County ◽

Nasopharyngeal Swab ◽

Suspected Case ◽

Full Genome ◽

Rt Pcr ◽

False Negatives ◽

Wide Range ◽

Local Transmission

LetterHere, I analyze the metagenome from the nasopharyngeal swab of a suspected case of local transmission of Covid-19, in Brazil (Accid:PRJNA613951).Very little viral load, not covering the entire genomeThere are just 152 reads matching to SARS-Cov2 [1] (SIbrazil/SARS-Cov2.reads.fa), which adds up a total of 21190 bp, much lesser than the 30000bp SARS-Cov2 genome. This is a very plausible cause for false negatives, there is just not enough virus to detect.For eg, if the RT-PCR test was looking for a genomic fragment in the spike protein (3879bp) from 369– 1174, it would not find a match. Thus, it is important to use multiple primers spread across the genome, something which the CDC test does not do.Much more bacterial load - Lautropia, Prevotella, Haemophilus dominatingThere are a wide range of bacteria (SIbrazil/allbact.list.txt,n=117) - Lautropia, Prevotella, Haemophilus dominating (Table 1). These are the same bacteria found in China [2] and San Diego county [3]. There are 10851 reads matching to bacteria (SIbrazil/allbactsequences.fa). These bacterial co-infections form the basis of hydroxychloroquine and azithromycin working in clinical trials [4].

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Pooled Saliva Specimens for SARS-CoV-2 Testing

Journal of Clinical Microbiology ◽

10.1128/jcm.02486-20 ◽

2020 ◽

Author(s):

Bidisha Barat ◽

Sanchita Das ◽

Valeria De Giorgi ◽

David K. Henderson ◽

Stacy Kopka ◽

...

Keyword(s):

Viral Load ◽

Screening Program ◽

High Viral Load ◽

Load Cycle ◽

Nasopharyngeal Swab ◽

Rt Pcr ◽

Viral Loads ◽

Viral Transport ◽

Average Loss ◽

Positive Agreement

We evaluated saliva (SAL) specimens for SARS-CoV-2 RT-PCR testing by comparison of 459 prospectively paired nasopharyngeal (NP) or mid-turbinate (MT) swabs from 449 individuals with the aim of using saliva for asymptomatic screening. Samples were collected in a drive-through car line for symptomatic individuals (N=380) and in the emergency department (ED) (N=69). The percent positive and negative agreement of saliva compared to nasopharyngeal swab were 81.1% (95% CI: 65.8% – 90.5%) and 99.8% (95% CI: 98.7% – 100%), respectively. The percent positive agreement increased to 90.0% (95% CI: 74.4% – 96.5%) when considering only samples with moderate to high viral load (Cycle threshold (Ct) for the NP <=34). Pools of five saliva specimens were also evaluated on three platforms: bioMérieux NucliSENS easyMAG with ABI 7500Fast (CDC assay), Hologic Panther Fusion®, and Roche COBAS® 6800. The average loss of signal upon pooling was 2-3 Ct values across the platforms. The sensitivity of detecting a positive specimen in a pool compared with testing individually was 94%, 90%, and 94% for CDC 2019-nCoV Real-Time RT-PCR, Panther Fusion® SARS-CoV-2 assay, and cobas® SARS-CoV-2 test respectively, with decreased sample detection trending with lower viral load. We conclude that although pooled saliva testing, as collected in this study, is not quite as sensitive as NP/MT testing, saliva testing is adequate to detect individuals with higher viral loads in an asymptomatic screening program, does not require swabs or viral transport media for collection, and may help to improve voluntary screening compliance for those individuals averse to various forms of nasal collections.

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