QIAstat-Dx Respiratory SARS-CoV-2 Panel Testing in Pooled Nasopharyngeal Specimens for COVID-19 Screening in a Low Prevalence Setting

Background: COVID-19 screening in healthcare facilities plays a key role in the management of the ongoing pandemic. Rapid and reliable detection of the virus ensures early identication of cases and targeted measures to prevent transmission of the virus. QIAstat-Dx Respiratory SARS-CoV-2 Panel (QRSP) is a fully automated rapid multiplex PCR assay for common respiratory pathogens including SARS-CoV-2 that can provide sample to result in 70 minutes. However, these tests are less suitable as screening tests because of their high cost and lower throughput. Objective: In this study, we evaluated the performance of QRSP on pooled nasopharyngeal specimens to reduce the cost and improve the turn-around time (TAT) for reporting negative COVID-19 results in a low prevalence setting. Methods: Nasopharyngeal (NP) specimens were simultaneously tested by pooled QRSP (~10 specimens/pool) approach and by standard RT-qPCR, and the results were compared. TAT of reporting negative results with pooled QRSP tests were compared to that of standard testing. Results: In 208 specimens, QRSP test results with specimen pooling were in 96% agreement (Kappa=0.92; 95%CI= 0.75-1) with standard RT-qPCR. Despite pooling, C T values obtained with QRSP were correlated with that of standard RT-qPCR (Pearson correlation coecient r=0.8343, p=0.0027). The median TAT for negative COVID-19 results by QRSP pooled approach was 2.8 hours (n=1305) compared to 5.4 hours by standard methods (n=4471). Conclusion: Pooled QRSP testing can be implemented for COVID-19 screening in low prevalence settings providing signicant cost savings and improving TAT without affecting test quality.


Introduction
Rapid testing to detect individuals infected with Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is central to the management of ongoing pandemic of coronavirus disease 2019 (COVID-19) (1).
Since the beginning of the outbreak, detection of viral RNA in nasopharyngeal (NP) swab specimens by real-time reverse transcription PCR (RT-qPCR) remains the main approach for identifying patients with acute infections (2). The choice of a method for SARS-CoV-2 RT-qPCR depends on multiple factors including required sample throughput, rate of positivity and the availability of resources. The World Health Organization (WHO) recommended pre-designed assays, targeting several SARS-CoV-2 genes, were made available for public use to enable the development of relatively inexpensive, laboratorydeveloped RT-qPCR tests. Typically, viral RNA from NP swabs is extracted and subjected to a screening PCR followed by con rmatory PCR of positive samples. Many commercial assays have also been developed in singleplex or multiplex formats to test for SARS-CoV-2 RNA. These tests are designed for high-complexity laboratories that perform large volume testing. However, these tests may have long turnaround time (TAT), and as such, pose problems when decisions about patient management and infection control are urgently required. Molecular testing devices that integrate RNA extraction and RT-qPCR with random-access features can signi cantly improve the TAT of COVID-19 testing (3).
QIAstat-Dx Respiratory SARS-CoV-2 Panel (QRSP) is a convenient, rapid and a fully automated solution for the detection of SARS-CoV-2 in near point-of-care (POC) settings. QRSP is a multiplexed RT-qPCR test for the detection of multiple respiratory pathogens, including the SARS-CoV-2 virus in nasopharyngeal specimens (4). While the test has the additional advantage of simultaneously detecting other respiratory viruses along with SARS-CoV-2, the test is more expensive than laboratory-developed assays and has low throughput as each machine can only test one sample at a time. To this end, the pooling of multiple specimens, in low prevalence settings, may signi cantly reduce the cost of the test and help report the negative results much faster, if the rate of positivity is low. In this study, we assessed whether pooling of up to 10 specimens affects the sensitivity of the test. QRSP pooled test results on 208 specimens were veri ed by individual tests by a WHO recommended, standard RT-qPCR. Our results suggest that positive and negative pool results by QRSP were in 95% agreement with that of standard method. Furthermore, we show that the use of QRSP pooled runs reduced the TAT of negative test reports by half.

Material And Methods
Evaluation of QRSP was performed in the Molecular Infectious Diseases Laboratory of Sidra Medicine, a 400-bed women's and children's hospital in Qatar, which was designated as a COVID-19-free facility, as part of an integrated, national pandemic management plan. Active screening of patients for COVID-19 was started in March 05, 2020. Standard COVID-19 testing in our laboratory involves extraction of viral RNA from nasopharyngeal ocked swab (NPFS)(BD) specimens in an automated nucleic acid extraction platform NucliSENS EasyMAG (bioMerieux) followed by RT-qPCR, based on one of the assays recommended by WHO (5). The performance standards of the standard method were established in our laboratory according to College of American Pathologists (CAP) guidelines. For QRSP testing, 0.1 ml of each of the 10 specimens were pooled together, vortexed for 10 sec and 0.3 ml of pooled specimen was analyzed by QRSP according to manufacturer's instructions (Qiagen).

Comparison of pooled QRSP tests with Standard SARS-CoV-2 RT-qPCR:
At the time of introduction of QIAstat-Dx for COVID-19 testing, on April 18, 2020, the prevalence of COVID-19 in our patient population was 0.44%. After initial veri cation of the QRSP assay, a total of 10 pooled runs (n = 105 specimens) that gave positive results and 14 pooled runs (n = 103 specimens) that gave negative results were individually assessed by standard RT-qPCR. Positive pool results were in 100% agreement with the standard assay. SARS-CoV-2 RNA was weakly (C T > 38) detected in only one of the specimen was found to be positive in each of the positive pools (Table 1). Overall agreement of QRSP pool results with standard RT-qPCR was 95% (Kappa = 0.9; 95%CI = 0.7-1.0). Also, the RT-qPCR C T values obtained by pooled QRSP test were positively correlated (Pearson correlation coe cient r = 0.8343, p = 0.0027) to that of standard RT-qPCR (Fig. 1).

Discussion
Since the emergence of COVID-19 outbreak, the WHO and many other health authorities around the world have emphasized the critical role of laboratory testing in case management, surveillance and rapid response, and infection prevention and control. In addition to the designated COVID-19 hospitals and testing centers, active surveillance is essential for hospitals that are intended to be kept COVID-19 free to ensure the safety of critical, non-COVID-19 related patients and the hospital staffs involved in the care of these patients. COVID-19 screening in these facilities is also necessary so that any patients, or their companions who test positive, can be immediately transferred to a COVID-19 facility. Rapid molecular tests results are crucial in this setting to reduce patient wait time in the emergency department waiting to be admitted or discharged. Rapid tests are also highly important for patients scheduled for surgery. Although the prevalence of the disease in this setting is very low, hospital laboratories are overwhelmed with the large volume of screening tests that cannot wait.
QRSP is the rst commercial rapid multiplex PCR assay for SARS-CoV-2 detection that has recently been independently evaluated and demonstrated to have high sensitivity and speci city against standard RT-qPCR tests. This test is easy to perform without any speci c special skills and is suitable for near pointof-care application (4

Conclusions
In conclusion, we evaluated a modi ed approach for COVID-19 testing by QRSP assay. We demonstrate that pooling of up to 10 specimens does not signi cantly affect the sensitivity of the assay, compared to standard RT-qPCR, but improve the TAT of negative test reporting. In order to save cost, and improve throughput and TAT of negative test results, pooled QRSP testing approach can be utilized for COVID-19 screening in low prevalence setting, in particular for screening in COVID-19-free hospitals or in health care facilities in countries where the number of cases are low or declining.