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Introduction
Since the spread of SARS-CoV-2 on 2019 to the end of the Public Health Emergency declaration realised by the WHO on May 20231-2, the number of confirmed cases reported reach 95,139.14 million around the world3. This, obviously, has meant a huge number of samples received by the different hospitals. The classical way to collect these samples is the use of swabs in universal transport medium (UTM) tubes, with no difference in the accuracy of viral detection between nasopharyngeal and oropharyngeal samples4. But the storage of this kind of samples can be a problem during emergency situations like during pandemic, when the great amount of them can saturate the diagnostic capacity of the laboratory, delaying the analysis of the samples, something that can affect the viral load on them by multiple factors5. So, the enlargement of storage and sample collection methods could be an interesting way to relieve pressure on points-of-care and other sample collection places. Based on an old method for sample collection, Guthrie cards, developed for their use to phenylketonuria detection by Guthrie test6, but also used for diagnosis of a small number of virus circulating in the blood7-10, this study explores their use for nasopharyngeal sample storage.

Material and Methods

• Samples: a set of 70 nasopharyngeal samples were collected in the Hospital Universitario Central de Asturias, on UTM collection tubes. Of the volume collected (1.5mL), one part (200μL) was extracted by the routine method on the lab, the MagNa Pure 96 system (Roche; Ginebra) and other part (100µL) was added to a clean Guthrie card and was left till total dry.

• Gutheriecars storage: after their total dry, this kind of samples were stored for 1 week at room temperature in a drower type card holder, using silica gel to avoid humidity.

• Gutherie card samples preparation: a circle of 1cm diameter of each dry Guthrie cards (ARCHIMEDlife; Vienna, Austria) sample was cut and incubated on 200µL of Minimal Essential Medium (MEM) (Dominique Dutscher, Brumath, France) with 50µL of proteinase K (pK) for 1 hour at 56ºC, to extract the viral particles from the cellulose matrix of the filter. After incubation, the total volume was extracted by the “Bikop” method, developed by our team, and exposed on previous articles11,12.

• Genome detection: all extracted samples from UTM tubes and Guthrie cards, were tested with a RT-qPCR, directed against different respiratory virus genomes, using 5µl of the sample, previously extracted by any of the tested methods. This volume was added to 6µl of TaqMan Fast 1-Step Master Mix (Life technologies, Carlsbad, CA) with 4µl of the adequate mixture of primers (Thermo Fisher Scientific, Walthman, MA) and taqman MGB probes (Applied Biosystems, Foster City, CA) (Table 1). Amplification and subsequent analysis were carried out using the Applied Biosystems 7500 Real-time PCR System (Applied Biosystems). The cycling protocol was, in all cases, as follows: (50ºC, 20 min; 95ºC, 5 min; 45 cycles of 95ºC, 10 sec; 55ºC, 15 sec and 60ªC, 30 sec).

Results

On the different samples analysed a variety of respiratory virus was searched, being detected the followed: SARS-CoV-2, Picornavirus, Respiratory Syncytial Virus (RSV) and Adenovirus (ADV). Detection results for all these viruses, including mean, range and IC 95% are shown on Table 2, exposed by cycle threshold and viral copies (log)/mL. Same data but for the SARS-CoV- 2 positive samples are shown on Table 3. On the Guthrie cards, the following samples for the 4 different viruses detected were: 33 (66%) for SARS-CoV-2, 6 (75%) for Picornavirus, 4 (100%) for RSV and 1 (50%) for ADV. When SARS-CoV-2 samples were analysed closely, the following samples were detected by cycle threshold ranges: 12 (60%) for Ct under 23, 12 (66.67%) for Ct between 23 and 29 and 9 (75%) for cycles over 30. On the other hand, when they were analysed by viral load, expressed as viral copies (log)/mL, the following samples were detected: 23 (62.16%) for viral loads over 5 log, 5 (71.43%) for viral loads between 4 anf 4.99 log, and 5 (83.33%) for viral loads under 4 log.

Discussion
Even when it is the classical and most recommended method for sample collection, specially when viral detection is involved, Universal Trasport Medium (UTM) tubes have a short expiration date after their use for sample collection. As Hosokawa-Muto et al (2014) have reported, different factors, like time, temperature and bacteria presence can affect the viral load collected, always decreasing it, being able to make it even undetectable5. Something than, as has been said before, can be a significant problem during special situations when work piles up. So, to propose an alternative for the storage this study analysed the use of Guthrie cards.

In general, after results obtained, we can affirm that a proper genome extraction of different virus can be performed from Guthrie cards, as we had obtained an amplification on 68.75% of the positive samples tested. Specifically, sensitivity for the different virus detected was: 33 (66%) for SARS-CoV-2, 6 (75%) for Picornavirus, 4 (100%) for RSV and 1 (50%) for ADV. The low number of samples of no SARS-CoV-2 virus detected represent a clear bias for the analysis, but the good results found by better analysing the SARS-CoV-2 results lead us to minimize it Focusing on these, which are the longest cohort of the samples, obviously for the actual situation, we see that 2 of each 3 extracted samples were detected after 1 week on Guthrie cards, with sensitivities upper 60% when we look the ranges analysed.

Moreover, when we consider ranges where the number of viral copies is lower (Ct ≥ 30 and log ≤ 3,99) we obtained good sensitivities (75% and 83.33% respectively). And, even when the number of positive samples for other virus is short, they show a similar tendency. On the other hand, results for SARS-CoV-2 show higher Ct/lower viral loads for Guthrie card samples than UTM for the ranges corresponding with the lowest amount of virus expected. This may have been caused by the concentration involved in the processing for the extraction of the Guthrie card samples, but not affect the results. It should  be said that the proposal to use this support as a genome bank was already made in 1994 by McEwen and Reilly14, in addition to considering the traditional use of this support for the detection of viruses present in the blood found in previous studies as: Human Immunodeficiency Virus (HIV), Herpes Simplex Virus (HSV), ADV or Citomegalovirus (CMV)7-10. So, with these facts and the results obtained, especially considering the time between preparation of samples on Guthrie cards and their processing for extraction and genome detection, something that has been validated before, we propose their use as a practical way of storage big numbers of viral samples, always taking care of humidity and temperature control.

Of course, more studies should be done to find the limits of this method, but now, it presents two great advantages over UTM tubes: one, their small size, so they can be storage in larger amounts, and two, the greater biodegradability of cellulose compared to the plastic UTM tubes15. On the other hand, their use for self-collection is a way that can be explored, as for this assay we start from collected nasopharyngeal samples, but sample collection on Guthrie cards will avoid the need for specialized workers and the massifications of sick people at the points-of-care for sample collection, or it can even be a solution when UTM are out of stock, as it was at the beginning of this pandemic, as American Society of Microbiology predicted16.

Conclusions
1. Guthrie cards are a viable method for nasopharyngeal samples storage.
2. Guthrie cards can storage different respiratory virus (SARS-CoV-2, Picornavirus, RSV and ADV) for at least a week.

Declarations
• FUNDING: Not applicable.
• Conflicts of interest / Competing interests: The authors declare no potential conflicts of interest or competing interests.
• Ethics approval: This study was approved by Comité de Ética de la Investigación del Principado de Asturias with code CEImPA 2021.188.
• AUTHORs cONTRIBUtions: All authors have contributed equally to this paper.
• Consent for PUBLIcation: All authors have expressed their consent for the publication of this paper. 
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