key: cord-0936460-6ye0al3q
authors: Barrera‐Avalos, Carlos; Luraschi, Roberto; Vallejos‐Vidal, Eva; Figueroa, Maximiliano; Arenillas, Esteban; Barría, Daniela; Hernández, Felipe; Mateluna, Carlos; Mena, Javier; Rioseco, Claudia; Torrent, Claudia; Vergara, Claudio; Gutiérrez, Gaby; Quiroz, Javiera; Alarcón, Javiera; Cartagena, Julio; Cayunao, Javiera; Mella‐Torres, Andrea; Santibañez, Álvaro; Tapia, Sebastián; Undurraga, Alejandro; Vargas, Deborah; Wong, Valentina; Inostroza‐Molina, Ailen; Valdés, Daniel; Imarai, Mónica; Acuña‐Castillo, Claudio; Reyes‐López, Felipe E.; Sandino, Ana M.
title: Analysis by real‐time PCR of five transport and conservation mediums of nasopharyngeal swab samples to COVID‐19 diagnosis in Santiago of Chile
date: 2021-11-15
journal: J Med Virol
DOI: 10.1002/jmv.27446
sha: feaa9708548719d88dfe92e30167dd34a7a2f654
doc_id: 936460
cord_uid: 6ye0al3q

Due to the COVID‐19 pandemic, many transport kits have been manufactured to preserve and transport nasopharyngeal swab samples (NPSs) from patients. However, there is no information on the performance of the different virus transport media (VTM) used in COVID‐19 diagnosis in the population of Santiago de Chile. We compared the RT‐qPCR amplification profile of five different viral transport kit mediums, including DNA/RNA Shield™, NAT, VTM‐N, Ezmedlab™, and phosphate‐buffered saline (PBS), for NPSs from Central Metropolitan Health Service, Santiago, Chile. The DNA/RNA Shield™ medium showed a better performance in terms of Cq and RFU values for the internal reference RNase P and viral ORF1ab probes. By contrast, the PBS transport medium registered higher Cq values for the viral and reference gene, compared to the other VTM. DNA/RNA Shield™ shows higher relative fluorescence units (RFUs) and lower Cq values for the reference gene. Collectively, our results suggest that the PBS medium could compromise the sample diagnosis because of its lower RT‐qPCR performance. The NAT, Ezmedlab and VTM‐N, and DNA/RNA Shield™ media show acceptable RT‐qPCR parameters and, consequently, seem suitable for use in COVID‐19 diagnosis.

K E Y W O R D S false negative diagnostic, nasopharyngeal sample transport kits, RNA subunit of ribonuclease P (RNase P) amplification, SARS-CoV-2 diagnostics, SARS-CoV-2 ORF1ab gene, virus transport medium

The severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) has led to a wide spectrum of challenges in the process of viral detection in the control of the COVID-19 pandemic.

The current massive worldwide molecular method to detect the SARS-CoV-2 genome in any sample is by RT-RT-qPCR, a molecular diagnostic technique that identifies the genetic material of the virus from the upper respiratory tract, including oropharyngeal, nasopharyngeal, or saliva samples. 1, 2 One of the critical steps for virus detection is the pre-analytical stage that involves the collection, preserving, and transporting of the sample to the clinical laboratory. 3, 4 The Centers for Disease Control and Prevention (CDC) indicates that the sample should be transported in a suitable VTM for an efficient diagnosis of the COVID-19. 5 On the other hand, the Food and Drug Administration (FDA) recommended the use of an alternative viral transport medium (VTM) to counteract the constantly increasing demand in the transport and preservation of the viral sample. 6 Owing to the stock-out suffered due to the constant increase in the demand for diagnosis of SARS-CoV-2, many transport kits have been manufactured and used to supply the minimum inputs necessary to maintain the traceability of the infection. However, it is necessary to determine the capacity of the different kits to preserve the SARS-CoV-2 virus for its correct diagnosis. In this study, we evaluated the performance of five commercial kits aimed for collection, preservation, and transport of nasopharyngeal swab samples (NPSs), for detection and diagnostic of SARS-CoV-2 by RT-RT-qPCR.

The solutions included in the analysis were DNA/RNA Shield™, NAT medium (NAT™), VTM-N, Ezmedlab™, and phosphate-buffered saline (PBS) transport medium. These solutions were chosen because they were available and widely used in the Central Metropolitan Health Service in Chile. We observed differences in the amplification of the internal control (RNase P) and viral gene (ORF1ab), probably associated with the different medium and preservation characteristics. Testing (Centers for Disease Control and Prevention. 7 In particular, the procedure was completed when the swab was saturated with fluid. Then, the NPSs were preserved and transported using: (1) Briefly, 250 µl of NPS from each patient was collected in a 1.5-ml tube and vortexed with 500 µl of lysis buffer (buffer RL: absolute ethanol; 1:1) for 1 min. Then, the solution was centrifuged at 14 000g for 5 min at room temperature. Subsequently, 700 µl of the lysate was transferred to a 96-filter plate and centrifuged at 1690g for 6 min. The 96-filter plate was washed two times with 400 µl of wash solution A. After each wash, the plate was centrifuged at 1690g for 4 min. Then, the plate was centrifuged at 1690g for 10 min to any volume trace. Finally, the total RNA was eluted using 70 µl of Elution solution A and centrifuged at 1690g for 7 min. The purified RNA was evaluated immediately by quantitative reverse transcription PCR (RT-qPCR).

The detection of viral SARS-CoV-2 genome sequence was carried out 

The sample size needed to achieve significant differences was calculated using G-Power Software (version 3.1.9.2). To do it, we applied the strategy of comparison of population means to determine significant differences between transport media kits. Thus, a medium effect size was set up (f = 0.25), with a α value = 0.05, and a power (1−β) = 0.8. Taking into consideration that there were five different experimental groups, we determined that an n total = 200 was required to find significant differences between different transport media. In our manuscript, the n total of samples analyzed was equal to 364. Thus, with this number of samples, we ensure the chance to find a significant effect. GraphPad Prism 8 statistical software was used to analyze and plot the data obtained. From the total received F I G U R E 1 RT-qPCR detection parameters for internal reference RNase P probe from nasopharyngeal swab samples (NPSs) preserved and transported in different viral transport media kits. (A) Cycle of quantification (Cq) and (B) relative fluorescent units (RFUs) value comparison for NPSs preserved and transported in DNA/RNA Shield™ (n = 136), NAT (n = 25), VTM-N (n = 90), Ezmedlab (n = 23) transport kits and PBS solution (n = 90), all followed by an RNA extraction process. For statistical analysis, one-way ANOVA-test with multiple comparison test and descriptive statistics analysis was applied. Lowercase letters above spot columns denote transport kits with no significant differences between them (p < 0.05). Below spots, the mean ± standard deviation (mean ± SD) is indicated from sample amplification parameters obtained for all kits evaluated samples from April to September 2020, the cumulative number of samples, frequency of age, and gender of the patient (by month in total and positive samples) were graphed. For comparisons between different transport media, the Cq and RFU values were analyzed. A one-way ANOVA was used to determine differences between the cycle of quantification (Cq) value and RFUs from samples preserved and transported in the mediums evaluated in this study. A p-value of <0.05 was considered statistically significant. , all followed by an RNA extraction process. For statistical analysis, one-way ANOVA test with multiple comparison test and descriptive statistics analysis was applied. Lowercase letters above spot columns denote transport kits with no significant differences between them (p < 0.05). Below spots, the mean ± standard deviation (mean ± SD) is indicated from sample amplification parameters obtained for all kits evaluated Service zone were preserved and transported routinely using the DNA/RNA Shield™ medium. However, because the stock-out suffered due to the constant increase in the demand for diagnosis of SARS-CoV-2 in Chile, we received NPSs in other four different transport mediums, including NAT and VTM-N, Ezmedlab transport medium, and PBS solution. The evaluation of these NPSs kits was made with the internal reference RNase P probe (that works as control of total RNA extraction) and the viral ORF1ab probe.

When the internal reference RNase P probe Cq value was compared between all transport mediums, they showed significant differences between them, except between NAT and Ezmedlab transport mediums ( Figure 1A) . 

The authors declare that there are no conflict of interests.

Conceptualization of the study was made by Mónica Imarai, Claudio 

All the information about the results is showed and described in this manuscript. The data used to support the findings of this study (S1 Data set) are available in Supporting Information.

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Analysis by real-time PCR of five transport and conservation mediums of nasopharyngeal swab samples to COVID-19 diagnosis in Santiago of Chile