By now, you have likely heard of the coronavirus outbreak. This most recent viral outbreak provides an example of how quickly a novel virus can spread throughout the world and how rapid in vitro diagnostic (IVD) development and deployment are critical to manage acute events like this.
On December 31, 2019 Chinese authorities reported a cluster of pneumonia cases in Wuhan City in the Hubei Province to the World Health Organization (WHO). On January 7, 2020 the cause was reported to be a novel coronavirus and given the scientific name Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and known globally to cause the Coronavirus Disease 2019 or Covid-19. Covid-19 was declared a Public Health Emergency of International Concern on January 30, 2020 by the WHO. The US Department of Health and Human Services (HHS) declared Covid-19 a public health emergency the next day (January 31, 2020).
The symptoms of Covid-19 are similar to the common cold and flu, including fever, dry cough and shortness of breath. The incubation period for Covid-19 varies widely from 1 to 14 days up to 24 days, and a mean of 5.2 days (WHO Interim guidance March 2, 2020). Some individuals remain asymptomatic. Together, these characteristics of the virus have made it difficult to determine whether an individual is infected. As a result, in a matter of months, confirmed cases have spread around the world. This outbreak has emphasized the need for quick global coordination in response and for the rapid development of vaccines, therapeutics and new IVDs.
Health organizations worldwide have responded. The WHO activated their R&D Blueprint procedures to accelerate R&D, improve global coordination, develop norms and standards and establish a response plan that includes surveillance, response and research. On February 29, 2020, the United States Federal Drug Administration (FDA) issued an Emergency Use Authorization (EUA), which allows the use of unapproved, potentially life-saving products during a public health emergency, meaning newly developed diagnostic tests can be used ahead of regulatory review and approval. These tests must be submitted to the FDA 15 days after validation.
SARS-CoV-2 is a betacoronavirus, similar to MERS-CoV identified in 2012 and SARS-CoV in 2002; its genetic material is single-stranded RNA. Coronaviruses are common in humans and are zoonotic (infect animals, e.g., camels, cattle, cat and bats). The initial diagnosis was based on genome sequencing. Release of the genetic sequence by Chinese authorities to the Global Initiative on Sharing All Influenza Data (GISAID) has allowed commercial entities to get involved in the development of subsequent real-time, reverse-transcription-polymerase chain reaction (rRT-PCR)-based diagnostics. The reverse-transcription step converts the viral RNA genome into cDNA (a complementary form of DNA), the PCR steps amplify specific genes within the viral genome and the real-time steps use fluorescence probes to measure gene products. To confirm whether a person is infected by Covid-19, it is recommended that two independent samples from the same individual demonstrate amplification of at least two SARS-CoV-2-specific gene targets.
On February 4, 2020, the US Center for Disease Control (CDC) was granted the first Emergency Use Authorization (EUA) of a diagnostic test for Covid-19. Unfortunately, the CDC test encountered several issues, including a flawed reagent (result of a design flaw or contamination) that caused inconclusive results (negative control looked positive) combined with too narrow testing inclusion criteria (requiring recent travel and symptoms), which delayed the widespread use of this test. The CDC 2019-nCoV Real-time RT-PCR Diagnostic Panel is a qualitative, molecular IVD that relies on nucleic acid amplification and increasing levels of fluorescence (real-time PCR arm) as an indicator of the presence of viral genes. The panel contains three primer/probes sets for three SARS-CoV-2 genes - one set is a universal SARS-like CoV gene and the other two primer/probes are specific for SARS-CoV-2. The kit also includes detection of the human RNase P gene to ensure proper experimental design and execution to confirm or detect: 1) the use of proper extraction methods, 2) the presence of human cell material, 3) any improper assay set-up or 4) a reagent or equipment malfunction. All three of the genes being tested need to be detected within 40 PCR cycles for the sample to be considered positive; fewer cycles than that result in an inconclusive test. Other commercial companies are joining the real-time RT-PCR-based diagnostic testing arena, including:
However, an issue with RT-PCR-based tests is that not everyone has the expertise and/or equipment to carry out these tests, so samples must be sent off to testing facilities, increasing the turn-around time (TAT) for a diagnosis, which is a critical issue in this acute phase.
Given this TAT issue, point-of-care serological tests, such as the one developed by Biomedomic (COVID-19 IgH-IgG Rapid Test) or WuXi Diagnostics (2019-nCoV IgM/IgG), that detect early and late antibodies against viral proteins from a blood sample are being developed. A Duke-NUS team identified antibodies that bind to the viral spike protein and acted to neutralize the viral infection in in vitro assays, and then they used synthetic viral proteins to create an ELISA to detect the presence of antibodies in blood samples (https://www.sciencemag.org/news/2020/02/singapore-claims-first-use-antibody-test-track-coronavirus-infections). However, nucleic acid detection remains the current gold standard and any positive serological test results are recommended to be confirmed using a follow-up PCR-based test. Serological tests help identify patients who may be asymptomatic or have already recovered from an infection and cleared the virus.
This example emphasizes the need for diagnostic testing companies, academic institutions and government agencies to work together to respond quickly to a global outbreak to develop IVD tests quickly that can inform, advise and control the spread of infectious diseases. Working together will hopefully bring accurate and precise tests with shorter TATs faster to the market in order to better control the spread.
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