Analytical Performance of COVID-19 Detection Methods (RT-PCR): Scientific and Societal Concerns

Abstract

Background. Health and social management of the SARS-CoV-2 epidemic, responsible for the COVID-19 disease, requires both screening tools and diagnostic procedures. Reliable screening tests aim at identifying (truely) infectious individuals that can spread the viral infection and therefore are essential for tracing and harnessing the epidemic diffusion. Instead, diagnostic tests should supplement clinical and radiological findings, thus helping in establishing the diagnosis. Several analytical assays, mostly using RT-PCR-based technologies, have become commercially available for healthcare workers and clinical laboratories. However, such tests showed some critical limitations, given that a relevant number of both false-positive and false-negative cases have been so far reported. Moreover, those analytical techniques demonstrated to be significantly influenced by pre-analytical biases, while the sensitivity showed a dramatic time dependency. Aim. Herein, we critically investigate limits and perspectives of currently available RT-PCR techniques, especially when referring to the required performances in providing reliable epidemiological and clinical information. Key Concepts. Current data cast doubt on the use of RT-PCR swabs as a screening procedure for tracing the evolution of the current SARS-COV-2 pandemic. Indeed, the huge number of both false-positive and false-negative results deprives the trustworthiness of decision making based on those data. Therefore, we should refine current available analytical tests to quickly identify individuals able to really transmit the virus, with the aim to control and prevent large outbreaks.

Keywords: COVID-19; RT-PCR; SARS-CoV-2; clinical pathology; false negative; false positive; pandemic.

Figure 1

Structure of SARS-CoV-2 virus. RNA of the viral genome is constituted by two genes—ORF1a and ORF1b—encoding for the nonstructural proteins NSP, while small genomic region includes S (spike protein), M (membrane protein), E (envelope protein), and N (nucleocapsid protein) genes. The S protein interacts with the angiotensin-converting enzyme 2 (ACE2) receptors of the host cell membrane through a receptor-binding domain (RBD) positioned on the spike protein. Once the ACE2/RBD interaction has been established, the virus can be endocytosed into the cell.

Figure 2

Estimated over time likelihood of SARS-CoV-2 virus detection with RT-PCR tests and serological assays in respect to symptom onset. The figure has been reconstructed on the basis of several reports and the estimated values are only approximate averages. Viral load shows an asymmetric distribution, with an extended tail. The maximum probability to detect infectious individuals happens after the first week since the infection and lasts for almost three weeks. Since then, it is quite unlikely to identify subjects that could truly transmit the disease. As a consequence, no virus development occurs in culture from biological samples in which RT-PCR positivity has been ascertained when the cycle threshold is very high (a Ct < 30 is suggested, but it may vary depending on the swab procedure accuracy). Unlike low-sensitivity methods (dashed line), the high sensitivity of the RT-PCR method could therefore lead to disclosing more positives that, on the other hand, are not contagious (solid line). Conversely, false-negative results may occur during the first week, when active viral replication does not reach the sensitivity level required by the RT-PCR test. For comparison, serological analysis shows a steady increase in both IgM and IgG values only after 4–5 weeks. Thereafter, IgM progressively decreases after 7–8 weeks, whereas IgG still persists for several months.