key: cord-354510-jlg5je0s authors: de Carvalho, A. F.; Goncalves, A. P.; Silva, T. B.; Sato, H. I.; Vuitika, L.; Bagno, F. F.; Sergio, S. A.; Figueiredo, M. M.; Rocha, R. P.; Fernandes, A. P. S.; Alves, P. A.; Teixeira, S. M.; da Fonseca, F. G. title: THE USE OF DENATURING SOLUTION AS COLLECTION AND TRANSPORT MEDIA TO IMPROVE SARS-COV-2 RNA DETECTION AND REDUCE INFECTION OF LABORATORY PERSONNEL date: 2020-06-20 journal: nan DOI: 10.1101/2020.06.18.20134304 sha: doc_id: 354510 cord_uid: jlg5je0s Background Since the emergence of the COVID-19, health officials have struggled to devise strategies to counteract the speed of the pandemic's spread across the globe. It became imperative to implement accurate diagnostic tests for the detection of SARS-CoV-2 RNA on respiratory samples. In many places, however, besides the limited availability of test reagents, laboratory personnel face the challenge of adapting their working routines to manipulate highly infective clinical samples. Here, we proposed the use of a virus-inactivating solution as part of a sample collection kit to decrease the infectious potential of the collected material without affecting the integrity of RNA samples used in diagnostic tests based on RT-qPCR. Methods Nasopharyngeal and oropharyngeal swab samples were collected from SARS-CoV-2-infected patients and from laboratory personnel using a commercially available viral transport solution (VTM) and the denaturing solution (DS) described here. RNA extracted from all samples was tested by RT-qPCR using probes for viral and human genes. Exposure of laboratory personnel to infective viruses was also accessed using ELISA tests. Findings The use of the DS did not interfere with the detection of viral genome or the endogenous human mRNA, since similar results were obtained from samples collected with VTM or DS. In addition, all tests of laboratory personnel for the presence of viral RNA and IgG antibodies against SARS-CoV-2 were negative. Interpretation The methodology described here provides a strategy that allow high diagnostic accuracy as well as safe manipulation of clinical samples by those involved with diagnostic procedures. Funding: CAPES, FAPEMIG, CNPq, MCTIC, FIOCRUZ and the UK Global Challenges Research Fund (GCRF). Background Since the emergence of the COVID-19, health officials have struggled to devise strategies to counteract the speed of the pandemic's spread across the globe. It became imperative to implement accurate diagnostic tests for the detection of SARS-CoV-2 RNA on respiratory samples. In many places, however, besides the limited availability of test reagents, laboratory personnel face the challenge of adapting their working routines to manipulate highly infective clinical samples. Here, we proposed the use of a virus-inactivating solution as part of a sample collection kit to decrease the infectious potential of the collected material without affecting the integrity of RNA samples used in diagnostic tests based on RT-qPCR. Nasopharyngeal and oropharyngeal swab samples were collected from SARS-CoV-2-infected patients and from laboratory personnel using a commercially available viral transport solution (VTM) and the denaturing solution (DS) described here. RNA extracted from all samples was tested by RT-qPCR using probes for viral and human genes. Exposure of laboratory personnel to infective viruses was also accessed using ELISA tests. The use of the DS did not interfere with the detection of viral genome or the endogenous human mRNA, since similar results were obtained from samples collected with VTM or DS. In addition, all tests of laboratory personnel for the presence of viral RNA and IgG antibodies against SARS-CoV-2 were negative. The methodology described here provides a strategy that allow high diagnostic accuracy as well as safe manipulation of clinical samples by those involved with diagnostic procedures. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) 1. In December 2019, Chinese health officials reported several cases of respiratory syndrome followed by pneumonia of unknown origin, initially in the city of Wuhan, capital of the Hubei province. The etiological agent behind the upsurge of the new syndrome was quickly identified as a new coronavirus, latter on named SARS-CoV-2. 1 The virus spread rapidly throughout China and in less than a month reached other countries in Asia, eventually reaching other continents. On March 11, 2020 , a global pandemic was declared by the World Health Organization (WHO). 2, 3 To date, more than 6 million cases and more than 440 thousand deaths due to the disease caused by SARS-CoV-2, named COVID-19, have been recorded worldwide, in 188 countries and territories around the planet with thousands of new cases and deaths been reported every day. 4 While health officials and governments around the world struggle to devise strategies to counteract the pace of the infection's spread, efforts to implement fast and sensitive approaches for diagnostic have emerged as key steps to control the epidemics. Real-time reverse transcriptase-PCR (RT-PCR) testing to detect SARS-CoV-2 RNA on samples collected from the largest possible fraction of the populations became an absolute consensus. 5 Widespread PCR testing has been pointed out as one of the most important elements in the successful COVID-19 containment strategy adopted by countries that have shown positive outcomes, including Taiwan, South Korea and Germany. 6, 7, 8 Nonetheless, having test kits available is not the only bottleneck to implement universal testing in many countries. The capability to adapt the routines of diagnostic laboratories to cope with the manipulation of highly infective clinical samples coming by the thousands is equally essential, especially considering that the RT-PCR diagnostic requires highly trained laboratory personnel. After peaking in Asiatic and European countries, the spread of the disease veered towards the Americas and possibly the sub-Saharan African continent. Developing countries in these continents may be hit hard by the pandemic for a number of reasons; and one particular troublesome aspect is the limited availability of diagnostic laboratories that will be able to cope with the huge incoming of clinical . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 20, 2020. . https://doi.org/10.1101/2020.06.18.20134304 doi: medRxiv preprint samples -either in terms of the total number of available laboratories, or their capability to deal safely with potentially infective clinical specimens. Therefore, the development of strategies to reduce the infectivity of clinical samples being sent to diagnostic laboratories could be essential to avoid contamination of the limited number of trained personnel and to maintain the operational capability of these laboratories. The high risk associated with biological samples determines that any clinical samples are to be considered as potentially infectious and, therefore, must be treated under strict biosafety protocols. 9 In this regard, national and international guidelines on biosafety concerning clinical laboratories must be followed in all circumstances. In the context of the current COVID-19 pandemic, there is still limited information regarding nosocomial infection by SARS-CoV-2 affecting health workers involved in diagnostics or similar activities. The WHO recommends that handling of clinical samples suspected of being infected with SARS-CoV-2 requires a BSL-2 or equivalent facility, whereas attempts to replicate the virus require at least BSL-3 facilities. 10 Several chemical and physical methods of viral inactivation have been proposed and evaluated for different pathogens, as a way to provide greater safety for professionals involved in the handling of potentially infectious samples and lower costs with laboratory infrastructure. 9, 11, 12, 13, 14, 15, 16 Amongst the chemical methods evaluated, the most commonly used products contain a chaotropic salt (guanidine), which acts as a denaturant agent for macromolecules culminating in virus inactivation. 12 At the same time, guanidine is able to decrease the degradation of RNA molecules in samples, acting as a ribonuclease inhibitor, therefore increasing the preservation of genetic material for application in molecular diagnostic methodologies in which RNA integrity is essential. 13 Here we describe the use of a simple, virus-inactivating and denaturing solution as part of a swab collection kit, aiming to decrease the infectious potential of the clinical sample and, at the same time, to preserve highly frail RNA molecules during transportation and short-term storage before testing. This low-cost, accessible . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 20, 2020. . https://doi.org/10.1101/2020.06.18.20134304 doi: medRxiv preprint approach has made it possible to achieve high diagnostic accuracy as well as manipulation safety for those involved with diagnostic procedures. The guanidine isothiocyanate solution used in this study is based on protocols established by Zolfaghari et al., 17 particularly concerned about the impacts of the intense flow of infective samples in a research laboratory that was adapted to join the testing effort with limited resources and personnel. In order to increase personnel safety, to avoid losing collaborators due to infections by SARS-CoV-2, and at the same time to increase preservation of the RNA contained in clinical samples, we introduced the use of the guanidinecontaining solution as collection and transport media instead of commonly used viral transport media (VTM). VTM is usually composed of a balanced salt buffer; sterile, heat-inactivated fetal bovine serum; and antibiotics, as suggested by the CDC. 20 As recommended in the WHO interim guidance protocol 10 , combined oropharyngeal and nasopharyngeal swabs were collected, using sterile flexible-rod swabs, and placed in . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 20, 2020. . a single sterile 15 mL polypropylene tube, containing 1.0 mL of the described denaturing solution. These collection kits were prepared in our laboratory and sent to hospitals according to their daily demand. Upon sample collection, the swabs remained immersed in the denaturing solution for at least 30 seconds, after which they were removed while being gently pressed against the tube wall to remove the excess absorbed solution. Swabs were, discarded in an appropriated biological waste disposal and were not sent to the diagnostic laboratory in order to minimize risks of personnel contamination. Clinical specimens from the laboratory personnel were collected multiple times and processed the same way as specimens from patients in hospitals (see below). Extraction of the total RNA from samples was performed using the QIAamp In order to verify the viability of the RNA stored in the denaturing solution (DS) proposed in this work, in comparison to the commonly used VTM, oropharyngeal and nasopharyngeal swab samples were collected from four laboratory's staff members and stored in VTM, following the protocol established by the WHO. Similarly, swab . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 20, 2020. . https://doi.org/10.1101/2020.06.18.20134304 doi: medRxiv preprint samples from the same staff were placed in the DS. After a two hours period, RNA extraction was performed from all samples, as described above, and the qPCR protocol was performed to detect the RNAse P gene. To evaluate for how long the DS could maintain viral RNA viable to be detected through qPCR, different DS tubes were spiked with 16400 copies of SRAS-CoV2 genomic RNA and maintained for up to 8 days at either 4 o C or room temperature. After that, the viral RNA was extracted and evaluated by qPCR. Results obtained were analyzed in the QuantStudio™ Design and Analysis software (v.1.5.1) and graphs were generated using the GraphPad Prism software (v.8.4.2). To evaluate the preservation of the viral RNA in VTM versus DS, clinical samples were collected from hospitalized COVID-19 suspected patients using either DS or VTM. Sample collection was carried out according to the WHO protocol and the molecular diagnosis was processed as described above. Obtained results were analyzed in the QuantStudio™ Design and Analysis software (v.1.5.1) and graphs were generated using the GraphPad Prism software (v.8.4.2). Having established that collection of clinical samples in DS preserves viral RNA in levels comparable to VTM, we opted to routinely receive and process only DS collected clinical samples, as described above. In order to assess the safety of our laboratory staff using such routine, we tested all laboratory personnel every 15 days. qPCR tests were conducted as described above. Additionally, serum samples from all laboratory members were also collected approximately 45 days after the beginning of the study, to assess the presence of antibodies against SARS-CoV-2. To that end, we employed an in-house anti-IgG COVID-19 enzyme-linked immunosorbent assay. Briefly, the nucleocapsid (N) protein of SARS-CoV-2 was expressed in transformed E. coli, purified, and used to coat 96 well ELISA plates. Tested sera were diluted in PBS-Tween20 solution, added to wells and incubated for . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. This work was approved by the UFMG's Ethics Committee and by the National Research Ethics' Committee, under number CAAE 31686320.0.000.5149. All laboratory personnel signed informed consents. Oropharyngeal and nasopharyngeal samples from four different laboratory members were collected in VTM or DS and processed. Viability of the extracted RNA was analyzed by qPCR, looking for the detection of the RNAse P human mRNA. We observed no differences in the cycle threshold (C T ) obtained for the detection of the targeted mRNA, regardless of the collection media (samples A to D), suggesting that the potential preservation of RNA in the two solutions is similar (Figure 1 ). . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 20, 2020. . Having established that DS can be reliably used to collect genetic material, we next asked for how long DS would keep viral genomic RNA viable for detection, either at . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 20, 2020. . . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 20, 2020. (Table 1) . PCR tests were also conducted using the Charité/Berlin protocol and results were similar (not shown). These findings indicate that there was no SARS-CoV-2 contamination of any of the professionals involved in the diagnostic process. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 20, 2020. 2 0 2 0 n d 2 5 , 7 2 5 0 5 / 1 5 / 2 0 2 0 n d 2 6 , 5 1 3 1 8 / 0 5 / 2 0 2 0 n d 2 6 , 8 2 1 1 3 / 0 5 / 2 0 2 0 0 , 1 5 2 n e g C T 0 0 1 0 3 / 2 3 / 2 0 2 0 n d 3 0 , 0 3 6 0 4 / 1 6 / 2 0 2 0 n d 2 1 , 7 9 6 0 4 / 2 8 / 2 0 2 0 n d 2 2 , 9 9 8 1 1 / 0 5 / 2 0 2 0 n d 2 4 , 0 0 4 0 5 / 1 4 / 2 0 2 0 0 , 0 9 7 n e g C T 0 3 8 0 3 / 2 4 / 2 0 2 0 n d 3 1 , 4 6 2 0 4 / 1 3 / 2 0 2 0 n d 2 6 , 7 2 7 0 4 / 3 0 / 2 0 2 0 n d 2 5 , 5 2 0 1 8 / 0 5 / 2 0 2 0 n d 2 3 , 8 4 3 0 5 / 1 3 / 2 0 2 0 0 , 1 7 8 n e g C T 0 3 6 0 3 / 2 5 / 2 0 2 0 n d 2 9 , 7 3 9 0 4 / 1 3 / 2 0 2 0 n d 2 6 , 7 4 8 0 4 / 3 0 / 2 0 2 0 n d 2 4 , 0 5 5 1 8 / 0 5 / 2 0 2 0 n d 2 5 , 5 8 8 0 5 / 1 3 / 2 0 2 0 0 , 2 0 3 n e g C T 0 0 3 0 3 / 2 3 / 2 0 2 0 n d 3 0 , 6 7 6 0 4 / 1 3 / 2 0 2 0 n d 2 8 , 8 6 1 0 5 / 1 1 / 2 0 2 0 n d 2 5 , 5 8 9 -n d -0 5 / 1 4 / 2 0 2 0 0 , 4 0 9 n e g C T 5 9 9 0 4 / 1 4 / 2 0 2 0 n d 2 7 , 9 0 5 0 4 / 2 8 / 2 0 2 0 n d 2 5 , 9 1 0 4 / 3 0 / 2 0 2 0 n d 2 4 , 7 1 5 1 6 / 0 5 / 2 0 2 0 n d 2 5 , 8 5 2 0 5 / 1 4 / 2 0 2 0 0 , 1 1 3 n e g C T 0 0 4 0 3 / 2 3 / 2 0 2 0 n d 3 3 , 7 6 1 0 4 / 0 2 / 2 0 2 0 n d 2 8 , 5 9 5 0 4 / 1 6 / 2 0 2 0 n d 2 4 , 1 1 4 3 0 / 0 4 / 2 0 2 0 n d 2 4 , 4 5 5 0 5 / 1 3 / 2 0 2 0 0 , 1 9 8 n e g C T 0 0 6 0 3 / 2 3 / 2 0 2 0 n d 3 2 , 1 4 9 0 4 / 1 3 / 2 0 2 0 n d 2 5 , 8 9 1 0 5 / 0 6 / 2 0 2 0 n d 2 7 , 6 3 7 1 6 / 0 5 / 2 0 2 0 n d 2 5 , 9 6 7 0 5 / 1 3 / 2 0 2 0 0 , 1 3 4 n e g M T 5 7 * 0 3 / 2 6 / 2 0 2 0 2 6 , 1 3 1 2 7 , 3 0 3 ----------3 , 4 7 4 p o s M T 3 5 * 0 3 / 2 3 / 2 0 2 0 2 3 , 4 6 3 2 7 , 9 5 1 ----------2 , 5 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 20, 2020. . The use of VTM is particularly indicated when virus viability is important, especially when SARS-CoV-2 isolation is to be attempted. However, only BSL-3 laboratories should be used to perform experiments involving replicative viruses, whereas diagnostic procedures that does not involve virus replication can be conducted in BSL-2 laboratories. 10 Media containing live viruses undoubtedly brings risks to laboratory personnel that manipulate clinical samples under lower biosafety standards. 28 Indeed, nosocomial exposition to SARS-CoV-2 in medical and laboratory personnel has been reported. 29, 30 Therefore, the use of collection and transport media able to inactivate SARS-CoV-2 with no loss of the diagnostic analytical power can be critical to avoid nosocomial infections in such laboratories. This is particularly desirable, as the pandemic epicenter is moving from Asia, Europe and North America to South America and Africa, where diagnostic laboratories with adequate biosafety structures are much scarcer. In addition, the use of a denaturing solution to collect and transport clinical samples, as the one described here, reduces costs in the processing of samples. 11, 12, 13 Our study have important limitations. First, this was not a case-control study, as our results were not compared to those obtained in a diagnostic laboratory routinely receiving clinical samples in VTM. Nonetheless, the differences in the possible extent of live virus exposition when VTM or DS are used are obvious. In this regard, the fact that none of our laboratory members was either infected or even seroconverted is an important indication that DS has been helpful in avoiding nosocomial exposition. Another limitation is that we are not able to quantify to which extent the good laboratory practices adopted in the laboratory could also be responsible for the verified results. Nonetheless, the use of the denaturing transport media is a critical part of such practices. Finally, we attempted to quantify the extent of SARS-CoV-2 inactivation using the described DS. To that end, isolated, laboratory cultivated live viruses were loaded on VTM or DS and plated on VERO cells. Viruses loaded on VTM remained replicative and able to generate plaques in the cellular monolayers (not shown). On the other hand, even when highly diluted, the guanidine salt present in DS was extremely toxic to cells, and the monolayers were destroyed before any eventual plaque could form. Nevertheless, it seems obvious to assume that, like cells, viruses were equally inactivated during exposition to DS. In spite of the limitations of our study, the use of denaturing, virus-inactivation solution as a . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 20, 2020. . https://doi.org/10.1101/2020.06.18.20134304 doi: medRxiv preprint collection and transportation media for diagnostic purposes is clearly an important asset to maximize clinical sample viability and minimize nosocomial infections in diagnostic laboratories, especially considering the SARS-CoV-2 spread to developing countries in which biosafe-structured laboratories are not easily available. We thank all personnel at UFMG's Vaccine Characteristics of and Important Lessons From the COVID-19) Outbreak in China: Summary of a Report of Cases From the Chinese Center for Disease Control and Prevention COVID-19) -Events as they happen. World Health Organization /events-as-they-happen>. 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