Comparison of Extraction Free Dry Swab with Standard Extraction-based RT-PCR Method for Diagnosis of SARS-CoV-2

INTRODUCTION

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative pathogen of the (COVID-19). In order to reduce the magnitude of the disease, measures like promoting quick diagnosis, early initiation of treatment, strengthening of quarantine facilities, and improvement in isolation have been advocated. The implementation of the above prevention and control measures can significantly aid in ensuring optimal containment of the viral infection.¹ False- negative/positive polymerase chain reaction (PCR) results must be ruled out by every laboratory and specific and accurate result must be provided so that the potential risk of transmission to household contact and community can be averted. This calls for the need that we must ensure appropriate sampling of specimens to facilitate the diagnosis of the novel viral infection.²

Collecting the appropriate specimen at the right time and transporting it to the laboratory under proper conditions are critical pre-analytical components of the testing process. The type of specimen collected will depend to some extent on the authorized SARS-CoV-2 viral test used. Such specimens may include healthcare personnel (HCP)-collected nasopharyngeal (NP), and oropharyngeal (OP) swabs.³ It should be made of synthetic fibers and have plastic or wire shafts. Besides this, there are new advisories by Indian Council of Medical Research (ICMR) suggestive of extraction-free protocol excluding VTM; dry swabs (DS).⁴ Considering the fact that the number of reported cases and the workload in the laboratory is extremely high, there have been issues with maintaining the supply chain and management, especially of the laboratory consumables. Moreover, we cannot rule out the shortage reported in the specimen collection sets and RT-PCR reagents.⁵ It is also important to note that a large number of national laboratories have been established that have been sharing the burden of laboratory consumables and other logistics.^6,7 Many kit producers eventually made their way into the expanding market with readily available kits. Due to the biomedical industry’s quick response and the expanding number of RT-PCR test kits available, there has been a gradual move towards utilizing conventional equipment to conduct RNA extraction.

Thus, the present study has been planned with the intention to assess the effectiveness of an extraction free dry swab (EFDS) protocol with extraction dependent viral transport medium (EDVTM) for the detection of the causative virus by RT-PCR; which has recently been used often to collect samples for the purpose of SARS-CoV-2 molecular detection. The effectiveness of the transport medium and dry swab was assessed by COVIPATH RT-PCR kit (Thermo Fisher Scientific). In this study, we have compared two methods on 500 clinical samples with EFDS and EDVTM protocol; as the reference method for diagnosis of SARS CoV-2 by RT-PCR.

MATERIALS AND METHODS

RESULTS

The flow and results of a performance characteristic of EFDS as compared to the reference method, EDVTM is as shown in Flowcharts 1 and 2.

The overall performance of, EFDS as compared with EDVTM showed; 500 samples were used, and 455 of them produced concordant results, while 45 samples produced discrepant findings (Table 1). Upon analysis, the positive and negative agreements were estimated as 85.65% (CI: 80.44 to 89.91%) and 98.47% (CI: 96.14 to 99.58%) respectively (Table 2). Among 500 samples, SARS CoV-2 was detected in 197 samples (39%) and not detected in 258 (51%) by both the assays; There were 33(6.6%) samples not detected by EDVTM but detected by EFDS method and 4 (0.8%) samples not detected by the EFDS method and detected by EDVTM, 8 samples showed inconclusive result that is only one gene positive (after repeating) of which 6 samples were positive for both genes in EFDS Method and 2 Samples were positive for both genes in EDVTM Method.

DISCUSSION

In order to control the spread of the infection, accurate identification of the infected individuals is the need of the hour. Given these various test results and the rapid spread of the infection, it is important that PCR should be widely practiced worldwide. Due to the ongoing pandemic, a massive number of swabs and PCR kits are required worldwide, placing a strain on resources and supply chains.⁸

The supply chains got significantly interrupted in the initial period of the pandemic itself, leaving laboratories struggling for any available commercial reserves. Several laboratories were driven to utilize different collection devices and transport media due to the pressure to enhance testing capacity, usually without conducting a standard test. Several scientists designed their own transport media because there weren’t any commercially accessible options.⁹ The existing protocol which was previously demonstrated to be acceptable was chosen because of the broad variety of specimens that can be transported.¹⁰

Collection and transportation of laboratory samples are executed with the use of alternative transport media.¹¹ These media are not typically thought of as being the best option for the preservation of samples. However, owing to the shortage of viral and universal transport media (UTM), there was widespread use of alternative such media, which may not be ideal for the detection of the causative virus.

Recently Indian Council of Medical Research (ICMR) issued an advisory, for the detection of the causative virus by real-time reverse-transcription PCR, the overall sensitivity of this study 85.65% is more than that of the ICMR advisory for dry swabs which is 79% sensitivity and there is a slight decrease in the specificity 98.47% as compared to advisory 99%.⁴

In our study, variability was reported between the EFDS method and the reference method for 45 discordant results. As per prior research, a variety of possible reasons could cause assay discrepancies.¹² Swabs collected in VTM are diluted with a VTM of 2 mL and the dry swabs are diluted with only 400 ul of buffers. There is five times more dilution of the viral load in VTM as compared with the EFDS method. Also, There are a variety of appropriate VTM formulations that might work for each laboratory’s specific set of conditions. While selecting a VTM formulation, the unique requirements of the shipping and receiving laboratories should be taken into account.

CONCLUSION

The EFDS assay showed a good correlation with the reference method. There was a slight increase in the detection rate showed by the EFDS assay as compared with EDVTM. As per these results; it can be concluded that EFDS method showed comparable performance with the comparative method and can be useful in for detection of SARS-CoV-2, EFDS method also reduces the cost and simplifies the workflow.

ACKNOWLEDGMENTS

Authors extend their regards to the colleagues who participated in this study. The study was sponsored by the Thyrocare Technologies Limited Team and Seven Hills Hospital.

ORCID

Prasad Tukaram Dhikale https://orcid.org/0000-0002-1860-6153

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