Fast, Reliable, and Simple Point-of-Care-like Adaptation of RT-qPCR for the Detection of SARS-CoV-2 for Use in Hospital Emergency Departments

Abstract

During COVID-19 pandemics, the availability of testing has often been a limiting factor during patient admissions into the hospital. To circumvent this problem, we adapted an existing diagnostic assay, Seegene Allplex SARS-CoV-2, into a point-of-care-style direct qPCR (POC dqPCR) assay and implemented it in the Emergency Department of Clinical Hospital Center Rijeka, Croatia. In a 4-month analysis, we tested over 10,000 patients and demonstrated that POC-dqPCR is robust and reliable and can be successfully implemented in emergency departments and similar near-patient settings and can be performed by medical personnel with little prior experience in qPCR.

Keywords: COVID-19; SARS-CoV-2; direct qPCR; dqPCR; emergency department; molecular diagnostics; point-of-care PCR; qPCR.

Figure 1

Scheme of the direct qPCR (dqPCR) method employed in this study. Nasopharyngeal and oropharyngeal swabs (1) were immersed in 2 mL of MG water (2), and samples were stored at +4 °C for a maximum of 5 h until further analysis. Within a Class II biosafety cabinet, samples were then vortexed for 20 s at high speed (3) and then left to rest for at least 1 min to minimize aerosol spread (4). Between 0.5–1 mL of each sample was then transferred into a separate qPCR grade microtube with a disposable Pasteur pipette (5). Viral particles were then inactivated by heating the samples at 70 °C for 10 min, and cellular debris was pelleted by centrifuging the samples in a tabletop microcentrifuge for 5 min at 1000× g. Following centrifugation, a small 10 µL sample aliquot was used directly in a qPCR reaction (8–10). Steps (3–7) and steps (8–10) were performed in physically separated laboratories to reduce the risk of sample cross-contamination. The figure was created with BioRender.com.

Figure 2

Sensitivity and specificity comparison of direct qPCR (dqPCR) and standard qPCR (stndqPCR). Only samples with valid control (IC) amplification were analyzed. (A) The patient structure included in the study: patients coming to the emergency department (ED) for initial COVID diagnostics (typical ER samples), convalescent patients (CRI) with confirmed infection with SARS-CoV-2 at least 2 weeks prior to this analysis, or patients hospitalized in the COVID respiratory center (CRC) of CHC Rijeka. (B) Diagnoses obtained by employing protocol recommended by the manufacturer with RNA isolation (stndqPCR) or direct qPCR (dqPCR) and utilizing criteria recommended by the manufacturer (Seegene criteria) or our criteria. The number of diagnoses per each protocol and criteria is shown inside bars. (C) Interrater agreement between diagnoses obtained with stndqPCR and dqPCR using kappa analysis utilizing two criteria for result interpretation. (D–F) Cycles of quantitation (Cq) for E gene (D), N gene (E), and RdRp/S gene (F) between stndqPCR and dqPCR. Upper panels depict samples from the same patient connected with the line. The bottom panels show violin plots of Cq values for all samples in the study. DL denotes detection limit in samples; above DL, no amplification was detected. Wilcoxon matched-pairs signed-rank test was used to determine whether Cq values differed significantly between groups, and statistical significance (p < 0.05) is labeled with *. (G) Pie charts showing the number of viral genes detected per test in two protocols and employing the two criteria for interpretation of the results. The number of patients with individual findings and corresponding percentage in the study are shown within each pie slice.

Figure 3

Sensitivity and specificity comparison of dqPCR and standard qPCR on typical ER samples. Only samples with valid internal (IC) gene amplification were analyzed. (A) Diagnoses obtained using a standard protocol with RNA isolation (stndqPCR) or direct qPCR (dqPCR) utilizing criteria recommended by the manufacturer (Seegene criteria) or our criteria. The number of diagnoses per each protocol and criteria is shown inside bars. (B) Interrater agreement between diagnoses obtained using stndqPCR and dqPCR and utilizing two criteria for result interpretation. (C–E) Cycles of quantitation (Cq) for E gene (C), N gene (D), and RdRp/S gene (E) between stndqPCR and dqPCR. The upper panels depict samples from the same patient connected with the line. The bottom panels show violin plots of Cq values for all samples in the study. DL denotes detection limit in samples; above DL, no amplification was detected. Wilcoxon matched-pairs signed-rank test was used to determine whether Cq values differed significantly between groups, and statistical significance (p < 0.05) is labeled with *. (F) Pie charts showing the number of viral genes detected per test in two protocols and employing the two criteria for interpretation of the results. The number of patients with individual findings and the percentage in the study are shown within each pie slice.

Figure 4

Application and validation of point-of-care (POC)-style dqPCR (POC dqPCR) in the ED CHC Rijeka. (A–E) Same samples from ED patients were analyzed in parallel utilizing POC dqPCR within the ED and dqPCR at the Center for Proteomics. (A) Diagnoses obtained by POC-dqPCR vs. dqPCR utilizing criteria recommended by the manufacturer (Seegene criteria) or our criteria. The number of diagnoses per protocol and criteria is shown in parentheses, where the first number is for POC dqPCR and the second for dqPCR. (B) Interrater agreement between diagnoses obtained using POC dqPCR and dqPCR utilizing two sets of criteria for result interpretation. (C–E) Cycles of quantitation (Cq) for E gene (C), N gene (D), and RdRp/S gene (E) between POC dqPCR and dqPCR. The upper panels depict samples from the same patient connected with the line. The bottom panels show violin plots of Cq values for all samples in the study. DL denotes detection limit in samples; above DL, no amplification was detected. Wilcoxon matched-pairs signed-rank test was used to determine whether Cq values differed significantly between groups, and statistical significance (p < 0.05) is labeled with *. (F) Pie charts showing the number of viral genes detected per test in two protocols and employing the two sets of criteria for result interpretation. The number of patients with individual findings and the percentage in the study are shown within each pie slice.

Figure 5

Sensitivity of dqPCR procedure in standardized samples (A,B) and applicability to pooled testing ©. (A) Validation of dqPCR method on standardized WHO-approved validation samples. Cq values of individual detected amplicons for stndPCR vs. dqPCR at different amounts of sample per reaction. (B) Diagnoses based on the results shown in A. (C) Titration curve using SARS-CoV-2 genome diluted in water or a negative pool sample and analyzed using dqPCR protocol. (D) Internal control (IC) amplification in reactions with different reference RNA concentrations diluted in either water or negative pool.

Figure 6

Application of POC-dqPCR in the ED setting—4 month analysis. First column shows distribution of results following first test. All invalid samples (no amplification in IC (SG criteria) or amplification ≤37 in our criteria) were retested from the same sample (second column). Monthly summary data (third column) shows data after invalid retest and added to the valid results of the first test. Wherever possible, presumptive (Seegene criteria) or inconclusive (our criteria) patients were re-tested by taking a new sample 24–72 h post first sampling (hPFS) unless these patients had a known prior COVID-19 infection (NR PP = not retested, known prior positive).