Sample pooling as a strategy for community monitoring for SARS-CoV-2

doi:10.21203/rs.3.rs-89650/v1

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Sample pooling as a strategy for community monitoring for SARS-CoV-2

https://doi.org/10.21203/rs.3.rs-89650/v1

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Sample pooling strategy was intended to determine the optimal parameters for group testing of pooled specimens for the detection of SARS-CoV-2 and process them without significant loss of test usability. Standard molecular diagnostic laboratory equipment and commercially available centrifugal filters, RNA isolation kits and SARS Cov2 PCR tests were used. The basic idea was to combine and concentrate several samples to the maximal volume, which can be extracted with the single extraction column. Out of 16 tested pools 12 were positive with cycle threshold (Ct) values within 0.5 and 3.01 Ct of the original individual specimens. The analysis of 112 specimens determined that 12 pools were positive followed by identification of 6 positive individual specimens among the 112 tested. This testing was accomplished with the use of 16 extractions/PCR tests, resulting in saving of 96 reactions but adding the 40 centrifugal filters. The present study demonstrated that pool testing can detect a single positive sample with Ct value as high as 34. According to the standard protocols, reagents and equipment, this pooling method can be applied easily in current clinical testing laboratories.

Virology

Infectious Diseases

Molecular Epidemiology

Covid-19

SARS-CoV-2

sample pooling

Real-Time RT-PCR

The coronavirus disease 2019 (COVID-19) pandemic has revealed the global concern of not only the ability to the appropriate management of COVID-19 disease, supporting the clinical decision-making process for infection control, but also survey of large asymptomatic populations.

Given the limited testing capacity available, it remains uncertain whether circulation of SARS-CoV-2 in the community precedes the identification of infected individuals. Rapid and accurate laboratory testing of SARS-CoV-2 is essential for early discovery, reporting, quarantine isolation, and cutting off epidemic transmission. Increasing of test number would enable to trace asymptomatic COVID-19 carriers and facilitate early detection of infection spread in community. Of particular importance is also to screen high risk populations (such as healthcare personnel, nursing homes), to estimate the effectiveness of community measures and social distancing and monitor a safe return to work, schools and universities. Efficient and higher capacity of laboratory diagnostics are needed to support such efforts.

Due to extensively validated standard operating procedure, the World Health Organization (WHO) recommended molecular reverse transcription PCR (RT-qPCR) testing of respiratory tract samples as method for the identification and laboratory confirmation of SARS-CoV-2 infection [1]. RT‐PCR assays targeting ORF 1a, ORF 1b, S gene, N gene of SARS‐CoV can detect <10 genome copies/reaction [2,3]. However, limitations on testing capacity have restricted the ability of governments and institutions to measure community prevalence, and minimize transmission. Sample pooling is a strategy used for community monitoring of other infectious diseases such as trachoma, tuberculosis [4,5]. While several pooling approaches for SARS-CoV-2 detection were recently reported [6-10], these studies mostly discussed theoretical considerations. In this study we demonstrated practical pooling solutions that save reagents and time by carrying out RNA extraction and RT-PCR on pooled samples.

Sample pooling strategy was intended to use standard molecular diagnostic laboratory equipment and commercially available RNA isolation kits and SARS Cov2 PCR tests. The pooling experiments were performed according to the instructions for particular tests from manufacturers. The underlying assumption was to pool multiple spesimens and process them with only one RNA isolation event and a single PCR test without significant loss of test sensitivity. The presented approach leads to effective cost drop up to six-fold per sample in comparison to individual patient testing. To test the effectiveness and sensitivity of that strategy, we performed two separate sets of experiments. In the first, one SARS CoV-2 positive sample was pooled with eight negative. In the second, one positive sample was merged with five SARS CoV-2 negative. The basic idea was to combine and concentrate several samples to the maximal volume, which can be extracted with the single QIAamp Mini column. Obtained Ct values for pooled samples were compared with Ct values for SARS CoV-2 positive samples processed individually. The difference between these Ct's reflects the possible loss of initial viral RNA templates number in merged samples.

Conducted experiments showed the limits of sensitivity of the proposed strategy of pooling samples and Z-Path-COVID-19-CE Genesig Real-Time RT-PCR test itself. The choice of that particular test was motivated by the extensive documentation prepared by the manufacturer. The kit's instruction contains such information as analytical sensitivity and specificity as well as repeatability, inter-instrument reproducibility, operator reproducibility, daily reproducibility and accuracy. The declared effectiveness of SARS CoV-2 RNA detection with a 100% replicate detection at the level of about five virus copies per reaction was also confirmed in our research. Seven tenfold dilutions of standardised SARS-CoV-2 RNA templates starting from 1,336x106 to 1,34 copies per reaction were amplified. The calculated standard curve showed excellent linearity r²=0,991, Figure 2.

All the swabs used in the project (both COVID 19 positive and negative) were processed (extracted and tested) individually to confirm their status. Also, all supernatants left after pooled samples concentrating were subject to further testing for SARS CoV-2 RNA presence, and no signal was detected.

To calculate the most efficient pool size a web-based application for pooling as described at https://www.chrisbilder.com/shiny was used. The hierarchical testing included an experimental prevalence rate of 5%, an assay lower limit of detection of 1 to 3 RNA copies/µL, an assay sensitivity of 97%, an assay specificity of 100%, a two-stage pooling algorithm, and a range of pool sizes of 3 to 20 samples. These calculations predicted the optimal testing configuration of an initial pool size of 5 followed by individual testing. The expected number of tests for the optimal testing configuration is 2.10. This leads to an expected number of tests per individual of 2.10 / 5 = 0.42. Thus, two-stage hierarchical testing reduces the expected number of tests by 58% when compared to individual testing. For the overall implementation of the algorithm, the sensitivity is 0.9409, the specificity is 1.0000, the positive predictive value is 1.0000, and the negative predictive value was 0.9969.

Six and nine of pooled samples were tested in two different experiments in eight different reaction sets. Results were summarised in Table 1 and Table 2.

Table 1. Six samples pooling tests results.

Sample name	Average Ct	∆Ct¹	Calculated starting templates quantity
pooled group #1	30.34 ± 0.42	1.57	169.82 ± 42.64
CoV-2 positive #1	28.78 ± 0.06		445.73 ±16.31
pooled group # 2	26.08±0.26	1.41	2459.16 ± 407.82
CoV-2 positive #2	24.68±0.06		5921.69±219.45
pooled group # 3	26.08±0.65	1.25	2562.31±898.64
CoV-2 positive #3	24.83±0.09		5383.47±295.71
pooled group # 4	31.36±0.14	0.50	87.74±7.68
CoV-2 positive #4	30.85±0.14		120.44±10.41
pooled group # 5	35.14±0.31	1.38	8.14±1.56
CoV-2 positive #5	33.76±0.32		19.47±4.06
pooled group # 6	-	-	-
CoV-2 positive #6	36.13±0.30		4.37±0.79
pooled group # 7	-	-	-
CoV-2 positive #7	36.06±0.16		4.52±0.47
pooled group # 8	31.58±0.45	1.86	78.09±22.88
CoV-2 positive #8	29.72±0.02		246.25±2.70
Average ∆Ct		1.33±0.46

¹ The difference between Ct values of the particular pooled group and corresponding positive sample.

Two of eight positive samples (#6 and #7) showed the Ct value below the Genesig Real-Time RT-PCR test guaranteed limit of detection, which is 4.64 viral copies per reaction, Table 1, Table 2. However, both samples were analysed six times, and the repeatability of the test results was 100%. Positive status of these samples could not be confirmed with two other COVID 19 IVD multiplex tests (data not shown).

Table 2. Nine samples pooling test results.

Sample name	Average Ct	∆Ct¹	Calculated starting templates quantity
pooled group #1	31.46±0.31	1.80	83.05±15.12
CoV-2 positive #1	29.66±0.16		256.22±24.97
pooled group # 2	27.34±0.24	2.62	1108.34±164.61
CoV-2 positive #2	24.72±0.01		5749.46±43.17
pooled group # 3	28.25±0.37	2.59	630.24±154.01
CoV-2 positive #3	25.67±0.05		3162.27±92.19
pooled group # 4	32.89±0.14	2.59	33.36±2.94
CoV-2 positive #4	30.29±0.18		171.58±19.55
pooled group # 5	37.30±1.72	3.01	2.69±2.45
CoV-2 positive #5	34.29±0.37		13.99±3.21
pooled group # 6	-	-	-
CoV-2 positive #6	36.80±0.06		2.83±0.11
pooled group # 7	-	-	-
CoV-2 positive #7	35.71±0.85		6.21±3.60
pooled group # 8	32.85±0.24	2.85	34.31±4.91
CoV-2 positive #8	30.00±0.14		205.49±17.99
	Average ∆Ct	2.58±0.42

¹ The difference between Ct values of the particular pooled group and corresponding positive sample.

Calculated average delta Ct value, showed a statistical difference between pooled and individual positive samples in examined sets. The average 1.33 cycles difference in six samples pooling, confirmed the 2.5 x loss of the initial amount of SARS CoV-2 RNA during the processing merged samples. The initial viral loss is even higher in nine samples pooling sets. The value of average delta Ct equal 2.58 can be translated to sixfold loss (5.97x) of the viral RNA through the pooling and concentrating process.

The analytical sensitivity of the Genesig Real-Time RT-PCR test is equal to 4.64 SARS-CoV-2 RNA copies in reaction. Basing on test's sensitivity and confirmed loss of RNA during the concentrating process, the minimal initial viral RNA templates number which could be detected is 11,6 viral copies in case of pooling of six samples, and 27.7 in nine samples merging. Many COVID 19 IVD multiplex real-time PCR test declare minimal analytical sensitivity on ten RNA copies in reaction. Pooling, concentrating and processing of six samples in single RNA extraction followed by SARS CoV-2 test, seemed to be an interesting alternative for lowering the already enormous COVID 19 molecular diagnostics costs.

This study demonstrates that new approach of specimens pooling can be easily provided in the laboratory to obtain faster and reliable test results. The primary and fundamental advantage of merging samples is the increase in availability and decrease in costs of high-tech molecular diagnostics. The most crucial disadvantage is lost of analytical sensitivity of the tests together with a growing number of pooled samples. The strategy presented in this paper showed that an acceptable balance between the quantity of simultaneously tested samples and the analytical sensitivity of SARS CoV-2 testing could be reached.

Sample pooling is a strategy already used in sample screening for human immunodeficiency viruses (HIV) [11], influenza [12] as a simple cost effective method to enhance the speed of diagnosis especially when large number of samples have to be tested during epidemic screening [13]. Due to the high sensitivity of the PCR test, the result should be positive if at least one of the samples is positive for SARS-CoV2. If the sample pool tests positive, each sample has to be re-tested individually. Recently, several research groups have proposed and developed testing protocols based on test pooling to make better use of the existing testing capacity [14-16]. For the purpose of test pooling, the sample swabs of test patients are used twice: once in an archive tube and once in the pool tube. Several samples are combined in the pool tube, so that they can be tested simultaneously.

In the present study we followed the testing protocols of RNA extraction and PCR not to change the sensitivity of the original test, because it is easy to carry out in the lab and thus less error-prone than tests where varying sensitivity has to be taken into account. We randomly pooled the patients samples in pools of 9 and 6 including one positive sample adding the step of samples concentration by centrifugation. We used positive samples with different load of viral RNA (Ct from 24.7 to 36.0) to validate the method for samples with small amount of viral RNA. We found that in pool size, in 2 positive sample pools a single sample with very low Ct value (Ct >35) were missed. Procedure processing of six samples in a single RNA extraction followed by the PCR seemed to be that equilibrium considering the mathematically calculated algorithm for pooled samples. The 2.5-fold loss of the initial amount of SARS CoV-2 RNA, which can be translated to the average 1.33 cycles difference between pooled and single processed sample is the disadvantage of the procedure. In the time of the pandemic, this seems to be an acceptable cost.

Three approaches to the pooling method can be applied: (i) collection of more than one swab to one sample with viral transport media; (ii) mixing of the samples from different patients prior to RNA extraction; (iii); separate RNA extraction from each sample and mixing many RNA isolates for one PCR test. Taking into account that, in the first option, when a pool turns out to be positive, repeat sample collection has to be done and that the third option is less economic, we made a choice for the second option with some modification with specimen concentration. The similar approach of pooling strategy, but without sample concentration step, was presented by Abdulhamid et al [14] who tested 1 positive sample pool and 4 negatives where maximum number of pooling of samples was assessed mathematically [14]. The results of that study showed that pooled specimens were positive within a range of 0 Ct to 5.03 Ct difference from the original samples. In our study, after samples were concentrated by centrifugation, the range of Ct differences was 0.5 to 3.01. The third pooling option was described by Gupta et al. [17] who recommended pooling of RNA samples before PCR as a faster and more economical method since otherwise when a pool turns out to be positive, repeat sample collection or RNA extraction has to be done [17]. Our results proved that the size of pooled specimens effectively diagnosed was equal to those pooled after RNA extraction the same being more cost-effective. In other published studies on pool testing the authors found that pooling of 1 positive sample up to a pool of 30 [14] or even 64 negative samples [6] can be done without affecting the results. We have to remember that testing capacity of too large pool size will be wasted due to the possible need of re-testing (when too many pools are tested positive). It is important to select a pool size that is optimal, in given the circumstances to make the best use of the existing testing capacity [18].

4.1. Sample collecting

At the Department of Virology with SARS Laboratory, Medical University of Lublin, Poland, samples from symptomatic patients, from the hospitals and samples from prospectively screened asymptomatic populations, such as hospital employees, and from the community were tested for SARS-CoV-2. In these samples, about 0.5% of SARS-CoV-2 tests were positive. According to the National Public Health guidelines, all samples were collected using a single swab for nasopharynx or combined deep nasal and oropharyngeal collection from the same patient. Nasopharyngeal swab samples were collected in 1ml Sigma Virocult® (Sigma-Aldrich). In the experiments, 8 SARS-Cov-2 positive and 104 negative patients swabs were used.

4.2. Samples concentration

The Amicon Ultra 0.5 ml Ultracell^® 30K centrifugal filters (Merck) were utilized to concentrate the swabs samples. Three samples of 140 microliters each were pipetted on a single filter. The obtained 420 microliters of fluid was spun to obtain the desired final volume (40º fixed angle rotor, 14,000 x g, room temperature). The exact time of centrifugation depended on the viscosity of the samples, pooling test's version and ranged from 10 to 25 minutes.

4.2.1. Nine samples pooling

One positive and eight negative individual nasopharyngeal swab samples, each collected in 1ml Sigma Virocult®, were first lysed under denaturing conditions, according to QIAamp Viral RNA Mini Handbook to inactivate RNases. The lysing buffer was replenished with the Genesig^®Easy RNA Internal extraction control, which was a part of Coronavirus COVID-19 Genesig^®Real-Time PCR assay. Then three Amicon Ultra 0.5 ml Ultracell^® 30K centrifugal filters were loaded with nine samples. The first filer was filled with 140 microliters of SARS CoV-2 positive material and two negative specimens 140 microliters each. Two next Amicon filters were topped up with triplets of negative samples (140 microliters each) to reach 420 microliters of capacity in both concentrating tubes. The set of filters were centrifuged to obtain a final volume of 46 to 47 microliters per filter. The samples concentrated on three separated Amicon filters were pooled. Received a volume of 140 microliters was used to isolate viral RNA in the single isolation, Figure 1a. The experiment was repeated on eight SARS CoV-2 positive and sixty-four SARS CoV-2 negative patient's swabs with three technical repeats.

4.2.2. Six samples pooling

The analogous procedure was adopted to concentrate and merge one SARS CoV-2 positive and five negative samples. Two Amicon Ultra 0.5 ml Ultracell® 30K centrifugal filters were loaded with 420 microliters of three pooled samples per filter. The time of spinning the filters was selected to obtain about 70 microliters of concentrated sample on each filter. Then samples were combined, and a total volume of 140 microliters was subject to RNA extraction, Figure 1b. The test was carried on eight SARS CoV-2 positive and forty SARS CoV-2 negative patient's swabs with three technical repeats.

4.3. RNA extraction and RT-PCR testing

The RNA of eight SARS CoV-2 patients and one hundred and four SARS CoV-2 negative was extracted from Sigma Virocult^® swabs (140 µl per swab) using QIAamp Viral RNA Mini Kit (Qiagen) and eluted in 60μL. The same protocol was adopted to isolate RNA from pooled samples. 8μL of RNA was used in 20μL reaction using Z-Path-COVID-19-CE Genesig Real-Time RT-PCR kit (Primerdesign). We followed kit instructions with thermocycler protocol: 1 cycle 55 °C 10 minutes; 1 cycle 95 °C 2 minutes; 45 cycles 95 °C 10 seconds; 60 °C 60 seconds. The quality of the RNA extraction step was verified by amplifying the Genesig^®Easy RNA internal extraction control. Bio-Rad CFX Connect™ Real-Time PCR detection system was used to complete all real-time PCRs. The detection format was as follow: Covid-19 in FAM (465-510) channel, and RNA internal extraction control in HEX (533-580) channel.

4.4. Amplification of standards and the standard curve calculation

The COVID-19 positive control template (PCT) was a part of the Z-Path-COVID-19-CE Genesig Real-Time RT-PCR kit and contained a standard number of copies of SARS-CoV-2 RNA specific sequence with a given concentration of 1,67x10⁵ copies per μl (1,336x10⁶ copies in reaction). The PCT was employed to the preparation of a series of 7 tenfold dilutions and calculation of the standard curve (Bio-Rad CFX Maestro software). The standard curve calculation was based on two separated experiments with five technical repeats per tested dilution.

4.5. Ethics statement

The study was approved by the Ethics Committee review board of Medical University of Lublin, Poland (KE-0254/104/2020).The patient informed consent was waived off by the Ethical committee as the research was done on the anonymised, de-identified RNA samples.

In our opinion, all ideas regarding SARS CoV-2 RNA pooling are worth developing to make the pandemic control more cost-effective. Moreover, this study showed that pooling approaches can facilitate patients testing during infectious disease outbreak mainly by expanding current screening capacities of detection both in the community and among healthcare providers. However, the crucial challenge of pooled specimens testing is the use of highly sensitive assays to avoid missing of low-positive samples.

Author Contributions: Conceptualization, R.S., I.K., A.S. and M.P.; methodology, R.S.; validation, R.S., I.K. and A.B.; formal analysis, R.S., I.K. and M.P.; investigation, R.S., I.K., A.S. and A.B.; data curation, R.S and I.K.; writing—original draft preparation, R.S. and I.K.; writing—review and editing, R.S., I.K. and M.P.; visualization, R.S.; funding acquisition, M.P. All authors have read and agreed to the published version of the manuscript.

Funding: This research received no external funding.

Conflicts of Interest: The authors declare no conflict of interest.

SARS-CoV-2	Severe Acute Respiratory Syndrome Coronavirus 2
Covid19	Coronavirus Disease 2019

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Sample pooling as a strategy for community monitoring for SARS-CoV-2

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