Received: 15 September 2021 | Accepted: 8 November 2021 DOI: 10.1002/jmv.27446 RESEARCH ARTICLE Analysis by real‐time PCR of five transport and conservation mediums of nasopharyngeal swab samples to COVID‐19 diagnosis in Santiago of Chile Carlos Barrera‐Avalos1 | Roberto Luraschi1 | Eva Vallejos‐Vidal1,2 | Maximiliano Figueroa1 | Esteban Arenillas1 | Daniela Barría1 | Felipe Hernández1 | Carlos Mateluna1 | Javier Mena1 | Claudia Rioseco1 | Claudia Torrent1 | Claudio Vergara1 | Gaby Gutiérrez1 | Javiera Quiroz1 | Javiera Alarcón1 | Julio Cartagena1 | Javiera Cayunao1 | Andrea Mella‐Torres1 | Álvaro Santibañez1 | Sebastián Tapia1 | Alejandro Undurraga1 | Deborah Vargas1 | Valentina Wong1 | Ailen Inostroza‐Molina1 | Daniel Valdés1,3 | Mónica Imarai1,3 | Claudio Acuña‐Castillo1,3 | Felipe E. Reyes‐López1,4,5 | Ana M. Sandino1,3 1 Centro de Biotecnología Acuícola, Facultad de Química y Biología, Universidad de Santiago de Chile, Santiago, Chile 2 Centro de Nanociencia y Nanotecnología CEDENNA, Universidad de Santiago de Chile, Santiago, Chile 3 Departamento de Biología, Facultad de Química y Biología, Universidad de Santiago de Chile, Santiago, Chile 4 Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Bellaterra, Spain 5 Facultad de Medicina Veterinaria y Agronomía, Universidad de Las Américas, Santiago, Chile Correspondence Felipe E. Reyes‐López and Ana M. Sandino, Abstract Laboratorio de Virología, Centro de Due to the COVID‐19 pandemic, many transport kits have been manufactured to pre- Biotecnología Acuícola, Departamento de Biología, Facultad de Química y Biología, serve and transport nasopharyngeal swab samples (NPSs) from patients. However, there Universidad de Santiago de Chile, Alameda is no information on the performance of the different virus transport media (VTM) used in Libertador Bernardo O'Higgins 3363, Estación Central, Correo 40, casilla 33, Santiago, Chile. COVID‐19 diagnosis in the population of Santiago de Chile. We compared the RT‐qPCR Email: felipe.reyes.l@usach.cl and amplification profile of five different viral transport kit mediums, including DNA/RNA ana.sandino@usach.cl Shield™, NAT, VTM‐N, Ezmedlab™, and phosphate‐buffered saline (PBS), for NPSs from Funding information Central Metropolitan Health Service, Santiago, Chile. The DNA/RNA Shield™ medium Ministry of Health, Goverment of Chile; Agencia Nacional de Investigación y Desarrollo; showed a better performance in terms of Cq and RFU values for the internal reference Departamento de Investigaciones Científicas y RNase P and viral ORF1ab probes. By contrast, the PBS transport medium registered Tecnológicas, Universidad de Santiago de Chile; Fondo Nacional de Desarrollo Científico higher Cq values for the viral and reference gene, compared to the other VTM. DNA/ y Tecnológico 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. Carlos Barrera‐Avalos and Roberto Luraschi contributed equally to this study. J Med Virol. 2021;1–8. wileyonlinelibrary.com/journal/jmv © 2021 Wiley Periodicals LLC | 1 2 | BARRERA‐AVALOS ET AL. KEYWORDS 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 1 | INTRODUCTION condition. The nasopharyngeal sample was taken by a trained healthcare worker and authorized for this procedure by the CMHS, The severe acute respiratory syndrome coronavirus type 2 following the indications detailed on the Interim Guidelines for (SARS‐CoV‐2) has led to a wide spectrum of challenges in the process Collecting and Handling of Clinical Specimens for COVID‐19 of viral detection in the control of the COVID‐19 pandemic. Testing (Centers for Disease Control and Prevention.7 In particular, The current massive worldwide molecular method to detect the the procedure was completed when the swab was saturated with SARS‐CoV‐2 genome in any sample is by RT‐RT‐qPCR, a molecular fluid. Then, the NPSs were preserved and transported using: (1) diagnostic technique that identifies the genetic material of the virus Genosur (catalog number: DM0001VR; Genosur LLC, NW) that from the upper respiratory tract, including oropharyngeal, naso- contains an RNA stabilization buffer called DNA/RNA Shield™ pharyngeal, or saliva samples.1,2 One of the critical steps for virus (Zymo Research Corp) (n total = 136 samples). (2) IMPROVIRAL™ detection is the pre‐analytical stage that involves the collection, NAT medium (NAT) (catalog number: 550040; Improve Medical preserving, and transporting of the sample to the clinical labora- Instruments Co. Ltd), medium based on guanidine salts (n total = 25 tory.3,4 The Centers for Disease Control and Prevention (CDC) in- samples). (3) Ezmedlab™ (catalog number: 04010206) medium dicates that the sample should be transported in a suitable VTM for based on guanidine salts (n total = 23 samples). (4) CITOSWAB® an efficient diagnosis of the COVID‐19.5 On the other hand, the Food VTM‐N medium based on guanidine salts (catalog number: 2118‐ and Drug Administration (FDA) recommended the use of an alter- 0015) (n total = 90 samples). (5) Winkler LTDA. PBS sterile solution native viral transport medium (VTM) to counteract the constantly (catalog number 634280) (n total = 90 samples). All the samples increasing demand in the transport and preservation of the viral arrived at the laboratory before the first 24 h after the sampling sample.6 Owing to the stock‐out suffered due to the constant in- collection taking care of the cold chain temperature. crease 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 2.2 | Total RNA extraction necessary to determine the capacity of the different kits to preserve the SARS‐CoV‐2 virus for its correct diagnosis. In this study, we Total RNA extraction was carried out using the Total RNA pur- evaluated the performance of five commercial kits aimed for collec- ification Kit (96 deep well plate format; Norgen Biotek Corp). tion, preservation, and transport of nasopharyngeal swab samples Briefly, 250 µl of NPS from each patient was collected in a 1.5‐ml (NPSs), for detection and diagnostic of SARS‐CoV‐2 by RT‐RT‐qPCR. tube and vortexed with 500 µl of lysis buffer (buffer RL: absolute The solutions included in the analysis were DNA/RNA Shield™, NAT ethanol; 1:1) for 1 min. Then, the solution was centrifuged at medium (NAT™), VTM‐N, Ezmedlab™, and phosphate‐buffered saline 14 000g for 5 min at room temperature. Subsequently, 700 µl of (PBS) transport medium. These solutions were chosen because they the lysate was transferred to a 96‐filter plate and centrifuged at were available and widely used in the Central Metropolitan Health 1690g for 6 min. The 96‐filter plate was washed two times with Service in Chile. We observed differences in the amplification of the 400 µl of wash solution A. After each wash, the plate was cen- internal control (RNase P) and viral gene (ORF1ab), probably asso- trifuged at 1690g for 4 min. Then, the plate was centrifuged at ciated with the different medium and preservation characteristics. 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 2 | MATERIALS AND METHODS quantitative reverse transcription PCR (RT‐qPCR). 2.1 | Samples 2.3 | SARS‐CoV‐2 detection by RT‐RT‐qPCR NPSs of patients that belong to the Central Metropolitan Health Service (CMHS; Santiago of Chile) were included in the study. The The detection of viral SARS‐CoV‐2 genome sequence was carried out swab samples were collected in patients who arrive at the Center using the ORF1ab probe (TaqMan™ 2019nCoV Assay Kit v1, Thermo with the suspicion of being infected with COVID‐19. At the time of Fisher Scientific, Cat. no. A47532) using a one‐step strategy. Positive sampling collection, none of the patients analyzed was already internal control probes for ORF1ab and RNase P (TaqMan™ admitted to the healthcare center for COVID‐19 or any other 2019‐nCoV Control Kit v1; Thermo Fisher Scientific, Cat. no. BARRERA‐AVALOS ET AL. | 3 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 A47533) were included and assessed individually in the 96‐well PCR 2.4 | Data representation and statistical analysis plate. The/polymerase from TaqMan™ Fast Virus 1‐Step Master Mix (Applied Biosystems™, Cat. no. 44‐444‐36) was included in each re- The sample size needed to achieve significant differences was cal- action. The reaction contained 5 µl of TaqMan™ Fast Virus 1‐Step culated using G‐Power Software (version 3.1.9.2). To do it, we ap- Master Mix 4X, 1 µl of ORF1ab assay 20X (FAM detector channel), plied the strategy of comparison of population means to determine 1 µl of RNase P assay 20X (HEX detector channel), 11 µl of nuclease‐ significant differences between transport media kits. Thus, a medium free water, and 2 µl of extracted RNA sample. The amplification effect size was set up (f = 0.25), with a α value = 0.05, and a power thermal conditions included the reverse transcription at 50°C for (1−β) = 0.8. Taking into consideration that there were five different 5 min, predenaturation at 95°C for 20 s, followed by 40 cycles at experimental groups, we determined that an n total = 200 was re- 95°C for 3 s and 60°C for 30 s. All the RT‐RT‐qPCR reactions were quired to find significant differences between different transport performed on the Agilent AriaMx Real‐Time PCR System (Agilent media. In our manuscript, the n total of samples analyzed was equal Technologies, Part no. G8830A). Quantification cycle (Cq) and re- to 364. Thus, with this number of samples, we ensure the chance to lative fluorescence units (RFUs) data were extracted from each NPS find a significant effect. GraphPad Prism 8 statistical software was using the Agilent AriaMx software. used to analyze and plot the data obtained. From the total received 4 | BARRERA‐AVALOS ET AL. F I G U R E 2 RT‐qPCR detection parameters for viral ORF1ab 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 = 61), NAT (n = 20), VTM‐N (n = 10), Ezmedlab (n = 23) transport kits and PBS solution (n = 19), 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 Metropolitan Health Service, Ministry of Health, Government of samples, frequency of age, and gender of the patient (by month in Chile (No. 370/2021), and following the Chilean law in force. Data total and positive samples) were graphed. For comparisons between analysis used for this study was conducted only using the internal different transport media, the Cq and RFU values were analyzed. A sample code numbers assigned at the moment to receive them for one‐way ANOVA was used to determine differences between the diagnostic purpose. Accordingly, the samples have been irreversible cycle of quantification (Cq) value and RFUs from samples preserved anonymized. and transported in the mediums evaluated in this study. A p‐value of <0.05 was considered statistically significant. 3 | RESULTS 2.5 | Ethics statement 3.1 | RT‐qPCR amplification profile of five different commercial transport media kits All the experimental procedures included in this study was authorized by the Ethical Committee of the University of Santiago of Chile (No. We analyzed NPSs collected in different viral transport mediums. 226/2021) and the Scientific Ethical Committee of the Central Most of the NPSs coming from the Central Metropolitan Health BARRERA‐AVALOS ET AL. | 5 Service zone were preserved and transported routinely using the viral ORF1ab probe mean RFU value of 7018 ± 1924 (Figure 2B). By DNA/RNA Shield™ medium. However, because the stock‐out suf- contrast, samples preserved and transported in NAT, VTM‐N, fered due to the constant increase in the demand for diagnosis of Ezmedlab mediums, and PBS solution showed no significant differ- SARS‐CoV‐2 in Chile, we received NPSs in other four different ences between them. 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 4 | D IS CU SS IO N control of total RNA extraction) and the viral ORF1ab probe. When the internal reference RNase P probe Cq value was The real‐time PCR (RT‐RT‐qPCR) technique has been the gold stan- compared between all transport mediums, they showed significant dard and the most recommended method for the diagnosis for differences between them, except between NAT and Ezmedlab Coronavirus SARS‐CoV‐2.8 One of the critical stages for its diagnosis transport mediums (Figure 1A). Samples preserved and transported in is the collection, preservation, and effective transport of the sample DNA/RNA Shield™ medium showed a Cq value 15.15 ± 1.71 to the laboratory of diagnostics due to the nature of the (Figure 1A). Samples preserved and transported in NAT and Ezmedlab RNA‐genome of the virus. However, the high demand for supplies mediums showed no significant differences between them for the during the COVID‐19 pandemic has generated several stock breaks internal reference RNase P probe. These samples showed a mean Cq of material and essential reagents. As consequence, currently, there is value of 17.02 ± 1.48 and 18.17 ± 1.72, respectively (Figure 1A). For a wide repertoire of kits for preserving and transporting the NPSs for the samples preserved and transported in VTM‐N medium and PBS the control of the COVID‐19 pandemic. However, the variation in the solution, there was a marked shift toward high Cq values, with the detection signal and Cq value observed in our study suggests that the mean Cq value of 23.32 ± 1.74 and 28.07 ± 2.37, respectively transport and preservation medium should be carefully considered (Figure 1A). for the detection of SARS‐CoV‐2 and its implications in diagnosis. In Concerning the RFUs value for the internal reference RNase P this line, previous studies have documented the efficacy of the use of probe, samples preserved and transported in DNA/RNA Shield™ different transport mediums for NPSs preservation, either commer- medium showed the highest mean RFU value, suggesting an im- cials9–12 or in‐house production13–15 for the detection and diagnosis proved quality of RNA extracted from NPSs. This transport medium of COVID‐19. Accordingly, Garnett et al.10 compared the perfor- showed a reference RNase P probe mean RFU value of 8665 ± 1731 mance of at least six swabs commonly found in primary healthcare (Figure 1B). On the other hand, samples preserved and transported in settings for disease diagnosis, showing no significant differences in NAT, VTM‐N, Ezmedlab mediums, and PBS solution showed no sig- viral detection. Thus, the results indicate that several alternatives for nificant differences between the RFU from internal reference RNase sample transport kits can be used if supplies run out. However, there P probe, with mean RFU values of 5554 ± 953.7, 5541 ± 612.4, are several other specimen transport and preservation mediums used 4903 ± 1000, and 5657 ± 1355, respectively (Figure 1B). extensively for the diagnosis of the disease in Santiago of Chile. To When the Cq value of the viral ORF1ab probe was compared date, they have not been studied or compared with each other. That between all mediums, these showed no significant differences be- is the case of the DNA/RNA Shield™, NAT, VTM‐N, Ezmedlab tween them, except for samples preserved and transported in PBS mediums, and PBS solution. In this study, the effect of different solution, where it shows higher Cq values compared to the other preservation and transport mediums for NPSs was analyzed and means of transport (Figure 2A). Samples preserved and transported in compared for the massive diagnosis of SARS‐CoV‐2 by RT‐qPCR. We DNA/RNA Shield™ medium showed viral ORF1ab probe mean Cq detected variations between these mediums in the Cq value for the value of 25.15 ± 6.28 (Figure 2A). This kit showed a wide range of amplification of the internal reference RNase P probe. In the case of viral Cq values as can be inferred from the standard deviation of its the viral ORF1ab probe, we detected no variations in the Cq value for value, with positive results from Cq 12.96 till 35.49, close to the limit the amplification, except for samples preserved in PBS solution. Re- of detection of the ORF1ab probe (Cq = 37.14) from Thermo Fisher garding the RFU values for internal reference RNase P and viral RT‐qPCR Kit. In addition, the Cq values observed for those samples ORF1ab probes, DNA/RNA Shield™ medium showed the highest transported in DNA/RNA Shield™ medium registered a greater RFU values when compared to the other mediums. number of samples with lower Cq value and closer to the limit of These differences between mediums may be associated with its detection compared to the other transport medium kits. composition in which the NPSs are preserved and transported. In the The samples preserved and transported in PBS solution showed first place, in the case of the DNA/RNA Shield™ medium its com- the highest viral ORF1ab mean Cq value of 30.27 ± 2.75 (Figure 2A), position stabilizes nucleic acids and biological samples at room tem- suggesting the transport medium could affect the quality of the RNA perature, in addition to completely inactivating viruses, bacteria, and extracted. fungi. This agent has been widely used for sample collection and Regarding the viral ORF1ab RFU value showed by these different transport of various infectious agents.16–18 Indeed, the group of mediums, samples preserved and transported in DNA/RNA Shield™ Hamilton et al.19 reported this reagent improves the detection of medium showed the highest mean RFU value, suggesting an im- SARS‐CoV‐2 in saliva samples, by detecting the Spike protein at proved quality of RNA extracted from NPSs. This medium showed a lower Cq values compared to samples preserved without DNA/RNA 6 | BARRERA‐AVALOS ET AL. Shield™. Similarly, the group of Coryell et al.20 reported that DNA/ heat and a potential lower RNA integrity for the sample, being easier RNA Shield™ medium gave the best results in terms of viral detec- its handling and processing in type 2 biosafety laboratories. This tion. Importantly, it also showed better stability compared to PBS study broadens the knowledge regarding the data obtained from solution after seven days of preservation. NPSs transport kits available in the market facing a stock‐out event in On the other hand, the NAT transport medium showed viral SARS‐CoV‐2 pandemic circumstances. These results also serve as ORF1ab Cq values similar to those observed with DNA/RNA Shield™ evidence for the application of these transport media in the massive Kit. However, NAT showed for the internal reference RNase P RFU diagnosis of new emerging infectious diseases, which facilitates the value about 3000 RFU less than samples preserved in DNA/RNA choice of the most viable sample transport kit. This is particularly Shield™ medium. The NAT medium consists of an RNA stabilization relevant in the context of respiratory viruses spreading to countries solution with guanidine salts. This is a strong chaotropic agent with where the laboratory's available massive infrastructure allows the the ability to deactivate viruses and preserve nucleic acids by dis- handling and processing of samples only in a class 2 biosafety abling the action of nucleases and protein structures.21 The use of condition. this viral stabilizing and inactivating agent has been widely used in the study of several viruses22,23 including SARS‐CoV‐2 for their detec- tion and diagnosis during the pandemic.12,24,25 For example, Carvalho 5 | CONCLUSION et al.26 analyzed NPSs kept in the medium with guanidine salts, ob- taining Cq values like those reported in our current study. On the Collectively, our results suggest that the DNA/RNA Shield™ medium other hand, the PBS‐preserved NPSs showed higher Cq values for shows a better performance in terms of Cq and RFU values for the internal reference RNase P probe than DNA/RNA Shield™, NAT, internal reference RNase P and viral ORF1ab probes. Therefore, its VTM‐N, and Ezmedlab mediums. This difference in the Cq values for use improves molecular diagnosis in relation to less ambiguity in the the internal control could be associated with the absence of a sta- results obtained. Samples kept and transported in PBS show worse bilizing solution capable of preserving the quality of the viral RNA in RT‐qPCR parameters than the other media tested. PBS does not transport kits containing only PBS medium. However, the use has contain any viral inactivator, so its prior thermal inactivation is been supported of PBS solution as alternatives to VTM medium for mandatory for handling in a type 2 biosafety laboratory. The NAT, SARS‐CoV‐2 testing27,28 even preserving the sample at room tem- Ezmedlab, and VTM‐N media show correct RT‐qPCR parameters and perature for 72 h10 or preserving the sample for a month at 4°C.29 are suitable for use in COVID‐19 diagnosis. The logistic difficulties for the primary healthcare centers to preserve the samples in PBS solution represent a serious disadvantage com- ACKNOWLEDGME NT S pared to other transport mediums because it is mandatory main- The Laboratory of Virology had support from the COVID‐19 diag- taining a cold chain temperature range from the sampling collection nosis in the University laboratories network (Ministry of Sciences, until processing. Furthermore, in the PBS‐based transport kits, there Ministry of Health, Government of Chile) for diagnosis tasks. A.S. was is a possibility for the potential presence of any remaining still active supported by the National doctorate scholarship no. 21180465 viral particle. Accordingly, prior heat inactivation is imperative for its (ANID; Government of Chile). The authors also thank the Rapid safe handling in laboratory conditions with a class 2 safety level. The Assignment of Resources for Research Projects on the Coronavirus handling of active SARS‐CoV‐2 samples of respiratory viruses is re- (COVID‐19; Project number COVID1038 (A.M.S and F.E.R.‐L.); ANID, commended under strict protocols of type 3 biological safety la- Government of Chile); Fondecyt regular (Project numbers 1201664 boratories.30,31 Therefore, in our study, the PBS‐preserved samples (M.I.) and 1211841 (F.E.R.‐L.); ANID, Government of Chile), and were necessarily thermally inactivated as described in the metho- DICYT‐USACH (Project number 021943AC (C.A‐C.) grants. EV‐V was dology section, where this process could affect the RNA integrity. In partially financed by FONDEQUIP grant (Project number this way, the information about the RFU value is essential for the EQM200016, ANID; Government of Chile). diagnosis, because samples with a low RFU are indicative of problems associated to sample degradation CONFLIC T OF INTERESTS This study expands the knowledge about the performance of The authors declare that there are no conflict of interests. NPS transport solutions in pandemic circumstances and indicates the kits available in the market for collection, maintaining, and trans- A UT H O R C O N T R I B U TI O NS porting samples and the modulation of Cq value range obtained for Conceptualization of the study was made by Mónica Imarai, Claudio the internal control and SARS‐CoV‐2 detection. In this way, the Acuña‐Castillo, Felipe E. Reyes‐López, and Ana M. Sandino; Daniel displacement of Cq toward higher values may compromise the sen- Valdés, Claudio Acuña‐Castillo, Felipe E. Reyes‐López, and Ana M. sitivity of the PCR for the diagnosis of SARS‐CoV‐2. This could in- Sandino supervised the study; Gaby Gutiérrez, Javiera Quiroz, Ailen crease the possibility to get potential false‐negative results. On the Inostroza‐Molina, Deborah Vargas, Daniel Valdés, and Claudio other hand, our results convincingly indicate that media containing Acuña‐Castillo were in charge of all the logistics of coordination and viral inactivators such as DNA/RNA Shield™ and guanidine salts give reception of samples from the Central Metropolitan Health Service; better parameters, also considering that it avoids viral inactivation by The logistics of sampling processing and RNA extractions were in BARRERA‐AVALOS ET AL. | 7 charge of Carlos Barrera‐Avalos, Esteban Arenillas, Daniela Barría, 11. Rogers AA, Baumann RE, Borillo GA, et al. Evaluation of transport Maximiliano Figueroa, Felipe Hernández, Carlos Mateluna, Javier media and specimen transport conditions for the detection of SARS‐CoV‐2 by use of real‐time reverse transcription‐PCR. J Clin Mena, Claudia Rioseco, Claudia Torrent, Claudio Vergara, and Microbiol. 2020;58(8):1‐5. doi:10.1128/JCM.00708-20 Valentina Wong; Roberto Luraschi, Javiera Alarcón, Julio Cartagena, 12. Bockel D, Van, Munier CML, Turville S, et al. Evaluation of com- Javiera Cayunao, Andrea Mella‐Torres, Álvaro Santibañez, Sebastián mercially available viral transport medium (VTM) for SARS‐CoV‐2 Tapia, and Alejandro Undurraga received the RNA extracted and inactivation and use in point‐of‐care (POC) testing. Viruses. 2020; 12(11):1‐11. doi:10.3390/v12111208 performed the RT‐qPCR reactions; Felipe E. Reyes‐López and Ana M. 13. Petersen J, Dalal S, Jhala D. Criticality of in‐house preparation of Sandino analyzed the data; Data representation was made by viral transport medium in times of shortage during COVID‐19 pan- Roberto Luraschi, Eva Vallejos‐Vidal, Claudio Acuña‐Castillo, and demic. Lab Med. 2020;52(2):e39‐e45. doi:10.1093/labmed/lmaa099 Felipe E. Reyes‐López; Carlos Barrera‐Avalos, Roberto Luraschi, Eva 14. Mcauley J, Fraser C, Paraskeva E, et al. Optimal preparation of SARS‑CoV‑2 viral transport medium for culture. Virol J. 2021;18(53): Vallejos‐Vidal, Claudio Acuña‐Castillo, Felipe E. Reyes‐López and Ana 1‐6. doi:10.1186/s12985-021-01525-z M. Sandino carried out the data processing and interpretation; Carlos 15. Smith KP, Cheng A, Chopelas A, et al. Large‐scale, in‐house Barrera‐Avalos, Roberto Luraschi, Eva Vallejos‐Vidal, Maximiliano production of viral transport media to support SARS‐CoV‐2 Figueroa, Mónica Imarai, Claudio Acuña‐Castillo and Felipe E. Reyes‐ PCR testing in a multihospital health care network during the López co‐wrote the original draft. All the authors provided critical COVID‐19 pandemic. J Clin Microbiol. 2020;58(8):1‐7. doi:10. 1128/JCM.00913-20 feedback and approved the final manuscript. All authors have read 16. Carrau L, Rezelj VV, Noval MG, et al. Chikungunya virus vaccine and agreed to the published version of the manuscript. candidates with decreased mutational robustness are attenuated in vivo and have compromised transmissibility. J Virol. 2019;93(18): D A TA A V A I L A B I L I T Y S T A T E M E N T 1‐20. doi:10.1128/JVI.00775-19 17. Birger R, Morita H, Comito D, et al. Asymptomatic shedding of re- All the information about the results is showed and described in this spiratory virus among an ambulatory population across seasons. Clin manuscript. 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