Severe social and economic damage from the 2019 coronavirus disease pandemic (COVID-19) has led to an increase in strategies to investigate the causative agent, severe acute respiratory coronavirus syndrome 2 (SARS-CoV-2).
A new study published as a preprint on bioRxiv* server, deals with the feasibility of using dry swab samples for virus isolation, compared to wet swabs, to reduce costs while improving security.
The need for study
Currently, most diagnostic tests depend on the quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) to detect viral ribonucleic acid (RNA), which is performed on nasopharyngeal or oropharyngeal samples in patients. However, this method is expensive, can yield results slowly, and has a significant false-negative rate.
These shortcomings have led to research into alternative virus detection techniques. This includes modifications to the PCR technique by removing the transport medium, for example, or omitting the RNA extraction step.
Previous research has pointed out that the cycle threshold (Ct) for PCR tests performed on dry swabs, ie those incubated in dry conditions and wet swabs, incubated in wet media. This has also, with the above changes, increased the possibility of reducing testing time and testing costs.
Upper airway swabs are also used to detect infectious virus particles. This is important because the presence of viral genomic fragments or even the entire genome is not an indicator of infectivity. Even a few weeks after the virus is cleared, viral nucleic acid may still be present in the host tissues. Therefore, isolation of live virus and successful culture is currently the only reliable evidence that a person is contagious at the time of testing.
Viral culture requires laboratory conditions for biosafety III, high-quality samples and is long-lasting. The costs of reagents and culture medium must also be taken into account. However, this is the closest approach to the gold standard SARS-CoV-2 infectivity test available so far.
The aim of the study
The aim of this study was to investigate the viability of SARS-CoV-2 for virus culture in dry swab samples. Viral stores of known titer were inoculated into certain inocula into swabs, just like samples collected from patients.
Swabs were collected in three groups to be processed separately. The first was a group stored in the form of dry swabs; the second contained swabs stored in the virus transport medium; and the last group contained swabs stored in buffer solution. They were then incubated at room temperature, 25 ° C, as well as 4 ° C.
Incubation was performed for 1, 4, 8, 12, 24, 48 and 72 hours, after which they were all maintained at -80 ° C until the next step. Prickly points were then used to infect Vero cells in culture.
The medium was then replaced with fresh. Uninfected Vero cells acted as cell controls, while cells infected with known virus stores were used as infection controls.
RNA extraction was then performed and RNA was detected by RT-qPCR. Discovered genes include ORF 1ab and nucleoprotein N.
What were the results?
Swabs in all three experimental groups yielded elue viruses that were viable to a similar extent both at room temperature and at 4oC. Ct values in all groups were comparable, in other words, for both ORF 1ab and N gene.
Ct values ranged between 16 and 19 after as much as 12 hours, at both temperatures, ie 25oC and 4oC.
Ct values slowly increased after 24 hours of storage, up to 72 hours, to between 24 and 36. This suggests a decrease in virus viability over time, so it is recommended that virus particles recover from experimental samples as early as possible.
However, the results also show that Ct values are lower by approximately 2 in viruses from the buffer storage buffer group, compared to the other two.
What are the implications?
“In this study, we show that viable viral isolates can be obtained and propagated by the dry swab sampling method.. “The use of dry swabs facilitates patient sampling and avoids the use of liquid transport media.
These features, in addition to no need to use virus transmission media and RNA extraction, make the testing process faster, cheaper and safer. Moreover, it would allow the cultivation of viable virus particles for different research purposes, as it shows that such particles can be obtained after incubation of a dry swab at room temperature or at a temperature of 4 ° C.oC.
* Important notice
bioRxiv publishes preliminary scientific reports that have not been reviewed and, therefore, should not be considered definitive, guide clinical practice / health-related behavior, or treat it as established information.