The heavy social and economic toll of the coronavirus disease 2019 (COVID-19) pandemic has led to upscaling of testing strategies for the causative pathogen, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

A new study, released as a preprint on the bioRxiv* server, deals with the feasibility of using dry swab samples to isolate the virus, as compared to wet swabs, to reduce costs while improving safety.

The need for the study

Currently, most diagnostic tests depend on the reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) to detect viral ribonucleic acid (RNA), conducted on nasopharyngeal or oropharyngeal samples from patients. However, this method is expensive, can be slow to produce results and has a significant false-negative rate.

These disadvantages have led to the exploration of alternative techniques for virus detection. This includes modifications of the PCR technique, by eliminating the transport medium, for example, or leaving out the RNA extraction step.

Earlier research has pointed out that the cycle threshold (Ct) for the PCR tests carried out on dry swabs, that is, those incubated in dry conditions, and wet swabs, incubated in wet media. This has also, with the above modifications, raised the possibility of reducing the testing time and the expense of testing.

Swabs from the upper airway are also used to detect infectious viral particles. This is important because the presence of viral genomic fragments or even the whole genome is not an indicator of infectivity. Even weeks after the virus is cleared, viral nucleic acid may still be present in the host tissues. This is why isolation of the live virus and successful culture is the only reliable evidence, so far, that the individual is infectious at the time of testing.

Viral culture requires biosafety laboratory III conditions, high-quality specimens and is time-consuming. The cost of reagents and culture medium must also be considered. However, this is the nearest approach to a gold standard test of SARS-CoV-2 infectivity available so far.

Study aim

The current study aims to explore the viability of SARS-CoV-2 for virus culture in dry swab samples. Viral stocks of known titer were inoculated in specific inocula into swabs, just like samples collected from patients.

The swabs were clustered in three groups to be processed separately. The first was the group stored as dry swabs; the second comprised swabs stored in virus transport medium; and the last group contained swabs stored in buffer solution. These were then incubated at room temperature, 25°C, as well as 4°C.

The incubation was carried out for 1, 4, 8, 12, 24, 48 and 72 hours, following which all were kept at -80°C until the next step. The spiked swabs were then used to infect Vero cells in culture.

The medium was then replaced with a fresh medium. Uninfected Vero cells acted as cell controls, while cells infected with known virus stock were used as infection controls, respectively.

RNA extraction was then carried out, and the RNA was detected using RT-qPCR. The genes detected included the ORF 1ab and nucleoprotein N.

What were the results?

The swabs in all three experimental groups yielded virus elutes that were viable to similar extents at both room temperature and 4^oC. The Ct values in all groups were comparable, in other words, for both the ORF 1ab and the N gene.

Ct values ranged between 16 and 19 after as long as 12 hours, at both temperatures, namely, at 25^oC and at 4^oC.

The Ct values slowly rose after 24 hours of storage, until 72 hours, to between 24 and 36. This suggests a decrease in viral viability over time, such that it is recommended to recover viral particles from the experimental samples as early as possible.

However, the results also show that the Ct values are lower by about 2 with virus from the buffered-storage group of swabs, compared to the other two.

What are the implications?

“In the present study, we demonstrate that viable viral isolates can be obtained and propagated from the dry swab sample collection method.” The use of dry swabs makes sampling of patients easier and avoids the use of liquid transport media.

These features, in addition to the absence of any need for the use of virus transport media, and for RNA extraction, render the process of testing faster, cheaper and safer. Moreover, it would allow viable virus particles to be cultured for various research aims, as it demonstrates that such particles can be obtained after incubating the dry swab at room temperature or at 4^oC.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Dr. Liji Thomas

Dr. Liji Thomas is an OB-GYN, who graduated from the Government Medical College, University of Calicut, Kerala, in 2001. Liji practiced as a full-time consultant in obstetrics/gynecology in a private hospital for a few years following her graduation. She has counseled hundreds of patients facing issues from pregnancy-related problems and infertility, and has been in charge of over 2,000 deliveries, striving always to achieve a normal delivery rather than operative.