In December 2019, several cases of atypical pneumonia were reported in the city of Wuhan in China. Laboratory tests confirmed that the causative pathogen was a novel coronavirus, provisionally named 2019-nCoV and now called SARS-CoV-2. SARS-CoV-2 causes the disease officially designated as COVID-19 by the World Health Organization (WHO). As of February 27, 2020, the WHO has recorded 82,294 cases of COVID-19 in 46 countries. Of these, 78,630 were reported in China and have resulted in 2,747 deaths.
On January 30, 2020, the WHO declared the COVID-19 outbreak a Public Health Emergency of International Concern. This designation makes reliable laboratory diagnosis critical to public health intervention efforts. “Our goal is early detection of new cases and to prevent further spread of the coronavirus,” said US Centers for Disease Control and Prevention (CDC) director Robert R. Redfield, MD.
With global spread of the virus threatening a COVID-19 pandemic, early detection can bolster vaccine and drug development. Here, we discuss how diagnostic tests for COVID-19 have evolved rapidly from early cases to the latest infections.
Early detection of an unknown virus
Early this year, researchers collected bronchoalveolar fluid samples from the first few patients and tested them for 22 viruses and bacteria using polymerase chain reaction (PCR). Previously identified coronavirus strains and other pathogens known to cause respiratory symptoms were not detected in the specimens. This was the first indication that the infectious agent was a novel, previously unidentified pathogen.
The team then isolated virus particles from the same patients and examined them using transmission electron microscopy. The virus showed distinctive spikes characteristic of coronaviruses. They used reverse transcriptase PCR (RT-PCR) to amplify a conserved region of betacoronaviruses, which yielded positive results. Betacoronaviruses have previously been implicated in outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS).
Finally, the researchers sequenced the viral genome. They performed sequence alignment and phylogenetic analysis of the novel virus genome using other betacoronavirus genomes (SARS-CoV and MERS-CoV) as a reference. There was less than 90 percent similarity in the conserved domains of the unknown virus and other betacoronavirus family members. Thus, the researchers concluded that the respiratory symptoms of patients were caused by a novel betacoronavirus.
Diagnostic tests at the forefront
During the weeks following the first reported cases, infectious disease researchers deposited SARS-CoV-2 genome sequences into open databases such as GenBank and GISAID. This spurred the international community of scientists and medical professionals to develop rapid and reliable molecular diagnostics.
Researchers at the Berlin Institute of Virology developed assays to distinguish SARS-CoV-2 infection from SARS-CoV based on the nucleotide sequence of the RNA-dependent RNA polymerase (RdRp) gene. They validated assay cross-reactivity using 297 clinical samples obtained from patients infected with known respiratory pathogens. A separate group at Hong Kong University developed a one-step quantitative RT-PCR assay to detect two different regions of the SARS-CoV-2 genome—open reading frame 1b (ORF1b) and the nucleocapsid (N) region. They validated the test in two patients with suspected COVID-19. The N gene assay was about 10 times more sensitive than the ORF1b assay in these clinical samples. Assays designed by both groups were shared with the WHO and dispatched for use in several countries.
In the US, the CDC developed a qualitative real time RT-PCR assay to detect SARS-like coronaviruses and to specifically identify SARS-CoV-2. The primers and probes were designed against the virus nucleocapsid region. Previously, samples obtained from individuals with suspected COVID-19 had to be sent to the CDC for testing. However, on February 4, 2020, the Food and Drug Administration (FDA) issued emergency use authorization of the CDC’s RT-PCR Diagnostic Panel. Now, the test can be shipped to US state and local public health laboratories, Department of Defense laboratories, and select international laboratories. “This continues to be an evolving situation and the ability to distribute this diagnostic test to qualified labs is a critical step forward in protecting the public health,” said FDA commissioner Stephen M. Hahn, MD.
*UPDATED MARCH 18, 2020: With over 3500 reported cases as of March 17, 2020, the US has revved up its COVID-19 diagnostic efforts. Earlier this month, the FDA granted EUA for a SARS-COV-2 RT-PCR Diagnostic Panel developed by the Wadsworth Center, housed within the New York State Department of Public Health. The test is to be performed on respiratory specimens collected from individuals who meet the CDC’s testing guidelines.
On March 12, Roche’s cobas SARS-CoV-2 test became the first commercial test to receive FDA EUA. The tests, designed to run simultaneously with other assays on the company’s cobas 6800/8800 Systems, can provide results in 3.5 hours. Roche has initiated shipment of 400,000 tests to hospitals and laboratories across the US.
On March 13, Thermo Fisher Scientific was also granted EUA for their COVID-19 test called the TaqPath COVID-19 Combo Kit. The kit identifies specific sequences in the SARS-CoV-2 ORF1ab, nucleocapsid-encoding region, and spike protein-encoding gene. The test is optimized for use on the Applied Biosystems 7500 Fast Dx Real-Time PCR instrument and provides results within 4 hours. The company had 1.4 million tests available to ship and expects to increase production to 2 million tests per week.
Other tests recently approved under FDA’s EUA include Quest Diagnostics’ SARS-CoV-2 Qualitative Real-Time RT-PCR, Quidel Corporation's Lyra SARS-CoV-2 Assay, LabCorp’s COVID-19 RT-PCR Test, and Hologic’s Panther Fusion SARS-CoV-2. These tests are approved for use in CLIA-certified laboratories that perform high-complexity tests.
On March 16, the FDA announced a regulatory change that allows states to authorize laboratories to develop their own COVID-19 diagnostic tests. Laboratories authorized by the state will no longer have to pursue EUA from the FDA. This will ensure tests are available to meet the rising public health demand.
Limitations of existing diagnostic tests
Although RT-PCR is a highly sensitive test, it may suffer some drawbacks. RT-PCR primers are generally designed against conserved regions of the SARS-CoV-2 viral genome. Coronaviruses have error-prone RNA-dependent RNA polymerases, making mutations and recombination events frequent. Mutations in SARS-CoV-2 are currently very limited. How the diagnostic specificity and sensitivity of RT-PCR-based assays may be affected if the mutation rate changes, needs to be considered.
Low viral loads in asymptomatic or mildly symptomatic patients may not be reliably detected by RT-PCR. While positive results are indicative of active SARS-CoV-2 infection, they do not rule out bacterial infection or co-infection with other viruses. Both positive and negative results need to be interpreted in combination with clinical symptoms and epidemiologic data.
Some state public health laboratories have encountered issues while validating the US CDC’s RT-PCR assay. They reported positive results for the assay’s negative controls. This produces an inconclusive result that impedes reliable clinical diagnosis. “…one of the reagents wasn’t performing consistently…redoing the manufacturing is the next step,” Nancy Messonnier, MD, director of CDC’s National Center for Immunization and Respiratory Diseases, said in a teleconference.
Currently, the diagnostic tests are performed on respiratory fluids (nasopharyngeal aspirates, bronchoalveolar lavage, and sputum) and serum. However, one study that used primers to amplify the SARS-CoV-2 spike (S) gene could not reliably detect the virus in non-respiratory body fluids such as blood and urine. The CDC is working to develop a blood test that will detect antibodies produced against SARS-CoV-2. This will aid better understanding of disease transmission and help detect infection in individuals with few or no symptoms.
Commercial diagnostics in development as of March 2, 2020
The US FDA has created an emergency use authorization review template that is available upon request to developers of diagnostic tests. To date, this template has been shared with more than 50 developers who have expressed interest in developing diagnostics for COVID-19.
The table below provides an overview of the latest development efforts underway globally.
|Amoy Diagnostics (Xiamen, China)||PCR-based SARS-CoV-2 detection kit has entered emergency approval by China’s National Medical Products Administration (NMPA)|
|altona Diagnostics (Hamburg, Germany)||Real time RT-PCR-based assay for qualitative detection of SARS-CoV-2 RNA in respiratory samples|
|Biomeme (Philadelphia, USA)||Portable Go-Strips run on the company’s mobile handheld qPCR device. Go-Strips have SARS-CoV-2 RNA targets (ORF1ab and S gene) multiplexed with Biomeme’s RNA extraction and RT-PCR control. Intended for research use only|
|Bioneer (Daejeon, South Korea)||Diagnostic test kit submitted for emergency use assessment and listing (EUAL) to the Korea Centers for Disease Control and Prevention (KCDC)|
|CerTest (Zaragoza, Spain)||Released two diagnostic kits. One amplifies a fragment of the S gene. The second identifies ORF1ab and N gene based on sequences published by the Chinese Center for Disease Control and Prevention|
|Co-Diagnostics (Salt Lake City, USA)||RT-PCR-based Logix Smart test amplifies conserved region in the SARS-CoV-2 genome. First US company to receive CE mark for COVID-19 diagnostic test|
|Coyote Bioscience (Beijing, China and San Jose, USA)||Prep Free qPCR assay provides results in one hour using the company’s portable qPCR station. Test being used for research use only in China, but submitted to NMPA for emergency authorization|
|GeneFirst (Oxford, UK)||Developing two test kits; a three-hour PCR-based test for SARS-CoV-2, and a multiplex PCR assay for SARS-CoV-2 and 17 other common viruses and bacteria|
|Genekam Biotechnology (Duisburg, Germany)||Singleplex and multiplex test kits to detect SARS-CoV-2 and distinguish it from other infections such as influenza, MERS, and viruses commonly found in bats|
|Genomica (Madrid, Spain)||Diagnostic kit to simultaneously analyze 96 samples within one hour. Seeking accelerated approval from the Spanish Agency of Medicines and Medical Devices|
|GenScript (Piscataway, USA)||One-step qRT-PCR detection assay that targets the RdRp, N gene, and E gene of the first SARS-CoV-2 sequence identified. Intended for research use only|
|IDbyDNA (Salt Lake City, USA)||Explify Respiratory test platform uses next generation sequencing-based metagenomic technology to detect SARS-CoV-2 and 900 other pathogens. Can differentiate SARS-CoV-2 from other coronaviruses, based on in silico validation|
|Kogene Biotech (Seoul, South Korea)||PowerChek2019 Real-time PCR Kit granted EUAL in Korea|
|LexaGene (Beverly, USA)||Open-access, fully automated LX Analyzer platform successfully detected SARS-CoV-2 RNA using the US CDC test|
|Novacyt (Paris, France and Camberley, UK)||The company's molecular division, Primerdesign, launched a qRT-PCR test that detects SARS-CoV-2 within two hours. Recently received CE marking. Seeks emergency use authorization from US FDA|
|PCL (Seoul, South Korea)||In the process of commercializing a multiplex diagnostic kit|
|Qiagen (Hilden, Germany)||QIAstat-Dx Respiratory 2019-nCoV Panel detects SARS-CoV-2 ORF1b and E genes, and differentiates it from 21 other respiratory syndrome pathogens. QIAstat-Dx platform is CE-marked but the test panel seeks emergency use authorization in US, Korea, and China|
|Roche (Basel, Switzerland)||Tib MolBiol’s LightMix RT-PCR kit detects RdRp, E gene, and N gene of SARS-CoV-2. The test is compatible with Roche’s Light Cycler 480 series and the Magna Pure24 instruments. For research use only|
|Seegene (Seoul, South Korea)||AllPlex assay kit simultaneously detects three SARS-CoV-2 target genes—RdRp, E gene, and N gene. Auto-analysis software provides test results within four hours. Received CE marking in Europe and emergency use approval in Korea|
|ThermoFisher Scientific (Waltham, USA)||RT-PCR test that runs on Applied Biosystems TaqMan Assay technology identifies sequences found in initial 44 SARS-CoV-2 genomes. For research use only|
|TCM Biosciences (Seoul, South Korea)||TCM-Q Corona III diagnostic kit seeks EUAL from KCDC|