key: cord-0903949-qkugsph8
authors: Estévez, Agustín; Catalán, Pilar; Alonso, Roberto; Marín, Mercedes; Bouza, Emilio; Muñoz, Patricia; Alcalá, Luís
title: Sample pooling is efficient in PCR testing of SARS-CoV-2; a study in 7,400 healthcare professionals
date: 2021-01-30
journal: Diagn Microbiol Infect Dis
DOI: 10.1016/j.diagmicrobio.2021.115330
sha: 94096635a3cafa5664f8d003e05a15632cd5349a
doc_id: 903949
cord_uid: qkugsph8

SARS-CoV-2 pandemic shows the importance of having efficient virus diagnosis, especially in groups of particular relevance such as healthcare professionals, without involving a large economic expense. This is a prevalence study carried out in 7,400 healthcare professionals in a 1,350-bed hospital in Madrid, Spain. Pools of 10 samples were performed, using the Xpert® Xpress SARS-CoV-2 test for the diagnosis from clinical samples of nasopharyngeal exudate. A previous study was performed to evaluate the effect of the dilution in terms of sensitivity. The estimated sensitivity was over 95%. 740 pools were performed, with a final result of 218 healthcare professionals being positive. Using the pooling system, the reagent cost reduction to the institution was 75.3%. It can be concluded that the described sample pooling system is a useful and efficient tool in the diagnosis of SARS-CoV-2 in certain groups, assuming a cost reduction without reducing the sensitivity.

The current SARS-CoV-2 pandemic has highlighted the need for a strong diagnostic capability of SARS-CoV-2 by healthcare hospitals. The most widely used technique in the diagnosis of SARS-CoV-2 is the detection of specific genes of the virus by polymerase chain reaction (PCR). The current available PCR methods require high technical capacity, providing results in several hours. For this reason, it is necessary to optimize a system that allows results to be obtained in a short period and in large numbers. Pooling systems for clinical samples have proven to be effective in terms of greater optimization of resources in obtaining results without a significant decrease in diagnostic accuracy in different infectious diseases [1, 2] . In the case of SARS-CoV-2 diagnosis, the pooling strategy has proven to be effective only as a tool to detect the virus at community level in circumstances of very low prevalence [3, 4] .

Our study aimed to assess the reliability and savings of a pooling system for PCR of nasopharyngeal samples for SARS-CoV-2 in healthcare workers with a moderate prevalence of the disease, using Xpert® Xpress SARS-CoV-2 (Xpert) test.

Ours is a 1,350-bed teaching institution serving an urban area of nearly 350,000 inhabitants in Madrid, Spain. More than 8,000 health professionals work in our hospital. The SARS-CoV-2 pandemic has led to a significant increase in the workload, resulting in a reconversion of resources to adapt to the new situation. During the Covid-19 outbreak, about 20% of our healthcare workers acquired the disease.

During the months of April and May 2020, a SARS-CoV-2 prevalence study was carried out on all of these personnel.

In this article, we analyse the data obtained with 7,400 healthcare professionals. The exclusion criteria to participate in the study were to present symptomatology compatible with COVID-19 at that time, or to have had a positive PCR for SARS-CoV-2 in the previous month. To the PCR determination, we used the Xpert® test, an effective technique with high sensitivity in the diagnosis of SARS-CoV-2, with a shorter response time than conventional PCRs and the advantage of being performed on direct sample [5] .

For the screening of SARS-CoV-2, we set up a pooling system of nasopharyngeal exudate samples. As a previous step to the development of the sample pooling system for the SARS-CoV-2 screening program in our hospital, we had the need to perform a preliminary evaluation to make sure that this system did not imply a significant loss of diagnostic sensitivity. For this purpose, we chose a series of SARS-CoV-2 positive clinical samples at different cycle threshold (Ct) values, and whose PCR had been performed by Xpert® test. We then evaluated the detection of these samples in a 1:10 dilution formed by the positive sample together with 9 other negatives. The data derived from this previous study are collected in the Results section.

Initially, we performed a primary pool of 10 samples [6, 7] with a volume of 35 microliters per sample, with a final volume of 350 microliters. Then, 300 microliters of the total pool volume are added to Xpert® test, which is processed by a GeneXpert® Infinity System. In the event of a negative test, we consider the total of 10 samples in the primary pool to be negative. In the case of positivity, we proceed to perform 3 secondary pools consisting of two pools of 3 samples and a third of 4 samples. The volume contributed by each of the samples in the secondary pool is 110 microlitres, adding 300 microlitres of total final volume to an Xpert® test.The samples belonging to a negative secondary pool are also considered as negative. In the case of a positive secondary pool, we proceed to perform an individualized Xpert® test for each sample belonging to that positive pool.

The resulting different data was incorporated into a Microsoft® Excel database for Windows and analysed using the IBM SPSS Statistics® software.

The Research Ethics Committee of the HGUGM approved the study protocol entitled "PROYECTO COVI-STATUS MARAÑÓN", version 7, on 29 May 2020.

During the study period, 7,400 healthcare professionals from our hospital were screened for SARS-CoV-2 using the pooling system described in the Methods section.

In order to estimate the diagnostic sensitivity of our sample pooling system, we carry out a pre-screening study. First, we selected 8 samples that had been individually analyzed by the Genexpert PCR system. These samples corresponded to patients in our hospital who had attended the emergency department the same week as this pooling system validation study was carried out. The samples were properly stored at a temperature of -80ºC. The Ct of the samples ranged from 28.4 to 41.4 for the E and N2 genes. We chose samples with elevated Cts to more realistically evaluate the robustness of the pooling system in maintaining its detection capability. Secondly, we diluted 35 microlitres of each of these positive samples together with 35 microlitres of 9 negative samples, in pools of 10 samples in total. Then, 300 microlitres of the total pool were added to a Genexpert cartridge and compared the detection and Cts of the samples tested individually versus testing them in a diluted manner in the pools of 10 (Table 1) . Regarding the PCR Ct for the genes analyzed, the median Ct for the E gene was 36 and for the N gene 39.5.

The Regarding the diagnosis of SARS-CoV-2 by sample pooling system, there is little published information on this subject.

The main limitations of the literature published so far are the small number of cases processed by this system or that it is based on mathematical models [9, 10] , and that there is no experience in the use of Xpert® test for PCR from sample pooling. In all cases, the PCR performed from the pooling, is a conventional PCR with previous RNA extraction [11, 12] , assuming a longer time to obtain results, an important limitation in the development of SARS-CoV-2 populationlevel screening programs. It is important to take into account the economic aspect when establishing SARS-CoV-2 diagnostic programs in large population groups. Early detection of potential transmitters of the virus is important in order to achieve appropriate social isolation of them and thus prevent the spread of the virus and the emergence of new outbreaks of the disease. However, such population-based screening programmes can be very costly to implement. The use of sample pooling systems such as the one proposed in this study and carried out in our care centre, is presented as an effective tool in those population groups where a low prevalence of the virus is suspected. In our case, the commercial house provides us with the individual Xpert® cartridge at an approximate price of 40€. If we had made individual determinations using this technique to each of the 7,400 workers in our hospital, the total cost would have been 296,000€, without taking into account the cost of hiring staff dedicated to processing the samples. Using the pooling system, the total number of cartridges needed was 1,828, assuming a total cost of 73,120€, which translates into a cost reduction of 75.3%. As for the personnel hired for the processing of the samples in the laboratory, it was two laboratory technicians and two microbiologists, divided into two morning and afternoon shifts. According to our calculations, the estimated workload by pooling system has been practically the same as the one estimated for the individual processing of the samples, not assuming an increase in the staff hired for the study.

As for the time of reporting of results through the pooling system, varies between one and three hours, depending on whether the sample is part of a negative primary pool or whether an individual determination is needed from a positive secondary pool. In this sense, an option to reduce response time would have been to perform individual test on all positive primary pools to avoid making secondary pools. However, the total number of tests that would have been done would be 2,180, almost 20% more tests and an extra cost of 14,080€.

Our GeneXpert Infinity System had a capacity of 24 cartridges and the average time for a test is 1 hour, the system was only capable of performing 24 tests per hour while with the pooling system that allows for a 75.3% reduction in the cartridges needed, it is estimated that it could perform an average of 24/(1-0.753)=97.2 tests per hour. Therefore, in a scenario where more than 24 samples are accumulated to be processed per hour and taking into account that the workload of pooling versus the traditional individualized system is similar, we could conclude that the response time of pooling would be less than the individualized system.

Moreover, assuming that there has been no need to increase the number of personnel hired, and that the reduction in the cost of reagent material for the performance of SARS-CoV-2 PCRs has been significant, we conclude that this is a cost-effective system. That translates into a reduction in the budget for the implementation of the screening program, without losing diagnostic sensitivity in a significant manner.

Regarding the limitations of our study, it is important to take into account that as with any pooling system, in which a dilution of the clinical samples is carried out, there is the possibility of reducing the diagnostic sensitivity [13, 14] .

However, our experience with the previous study, in which we added a positive sample with a late amplification cycle (higher in some cases than the 42 CT) within a pooling of 10 samples made up of that positive sample and another 9

negative ones, confirmed the detection of that positive sample within the pooling in almost 100% of the cases. For this reason, we assume that the balance between potential loss of sensitivity and the high performance of the technique in terms of cost savings and efficiency is positive.

In view of our results, we conclude that the sample pooling system is an effective method for population-based screening for SARS-CoV-2 in groups where the prevalence of infection is low. It represents a considerable cost reduction without significantly increasing the workload in the laboratory and without reducing diagnostic sensitivity. It is a method that is easy to implement in conventional clinical microbiology laboratories. The Xpert® test system has high diagnostic sensitivity, and its processing characteristics from direct sample, integrated RNA extraction and subsequent PCR in less than an hour, make it a technique of choice in this type of program, due to its speed and reliability.

Finally, we believe it is necessary to carry out new studies using this methodology with the aim of implementing it in the future as a tool for the population diagnosis of SARS-CoV-2 in certain selected groups.

conceptualization, data curation, formal analysis, methodology. L.A.: conceptualization, data curation, formal analysis, methodology, writing-review and editing. Funding: This work was supported by the private company ADVENT INTERNATIONAL ADVISORY, S.L.U. who made a contribution of 180,000€ for this study. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication

Álvarez-Uría (Ana)

Olmedo (María)

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