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Coronavirus and the race to distribute reliable diagnostics

International teams worked at speed to make tests for the virus available in record time.
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The medical community is rallying to develop a set of rapid and reliable molecular diagnostic tests for the new human coronavirus that appeared in China — now dubbed sudden acute respiratory syndrome coronavirus-2 (SARS-CoV-2).

Coronaviruses are so called because the projections that encircle the capsid resemble a monarch’s crown when viewed under a microscope.BSIP SA / Alamy Stock Photo

This follows a breakneck effort by several research groups to identify and sequence the virus, sharing several viral genomes in open databases over recent weeks. As infections continue to mount — 74,185 people were reported to be infected across China as of midnight local time on 18 February — and authorities seek to accurately diagnose and document infections, the need for rapid and reliable diagnostics increases. Although many clinics still lack access to a robust, accurate and sensitive SARS-CoV-2 test, impressive progress has been made in a matter of weeks. In contrast, it took almost six months to identify and to establish assays for the coronavirus responsible for the 2002–2003 SARS outbreak.

After clusters of pneumonia cases of no known cause were epidemiologically linked to a seafood and ‘wet’ animal wholesale food market in Wuhan, China, three groups worked tirelessly to identify the culprit. First, researchers from Shanghai, Wuhan, Beijing and Sydney used metagenomic RNA sequencing to identify the previously unknown coronavirus in a sample from a single patient who had worked at the market. This team deposited a draft genome sequence in the publicly available GenBank sequence repository on 10 January; the current version was deposited on 17 January.

Around the same time, a rapid-response team dispatched by the Chinese Center for Disease Control and Prevention reported that they had isolated and cultivated a novel coronavirus from bronchoalveolar lavage fluid of three patients and identified it as the probable source of the outbreak. Electron microscopy observations uncovered a typical coronavirus morphology; light microscopy work demonstrated it had cytopathic effects on human airway epithelial cells. Genome sequencing revealed the virus shared over 85% sequence identity with a known SARS-like coronavirus found in bats. On 12 January, this group deposited three more genome sequences in the open access database Global Initiative on Sharing All Influenza Data (GISAID).

A third group of scientists, located in Wuhan and Beijing, identified and characterized the same virus in a further five patients with severe pneumonia and deposited five more genomes with GISAID. They also showed that the virus uses angiotensin-converting enzyme II (ACE2) to gain entry to host cells, as did SARS-CoV, the strain that caused the 2002–2003 outbreak, which infected 8,096 people and caused 774 deaths.

Several international groups have been working from these sequence data to design primers for polymerase chain reaction (PCR) tests to support global public health laboratories in the absence of a commercial test for SARS-CoV-2 (formerly 2019-nCoV). The lab of Christian Drosten, of the Institute of Virology, Charité University Hospital, Berlin, along with academic collaborators in Europe and Hong Kong, published details of a real-time PCR (RT-PCR) diagnostic test and workflow on 23 January, which detects SARS-CoV-2 and distinguishes it from SARS-CoV. The group verified the test in the absence of SARS-CoV-2 isolates or patient samples but confirmed its specificity against 297 clinical samples from patients with various other respiratory infections. This formed the basis of shipments of 250,000 kits, which the World Health Organization (WHO) dispatched to 159 laboratories across the globe in recent weeks.

Meanwhile, a group at Hong Kong University have developed two one-step quantitative RT reverse transcription PCR tests targeting both the open reading frame 1b (ORF1b) and the N regions of the viral genome based on the first sequence deposited at GenBank; these two test have been validated using two clinical specimens obtained from patients infected with SARS-CoV-2. The tests are explicitly designed to identify multiple viruses in the sarbecovirus subgenus to which SARS-CoV-2 belongs, given a lack of data on the genetic diversity of SARS-CoV-2 in humans and animals. As no other sarbecoviruses are known to be circulating in humans, a positive test can be considered as confirmation that a subject is infected with SARS-CoV-2 or a related animal virus. The N gene assay is recommended as a screening test and the ORF1b test is recommended as a confirmatory test. The diagnostic algorithm is similar to that followed for the Middle East respiratory syndrome (MERS) coronavirus, which was first identified in Saudi Arabia in 2012. “We basically just tried to adopt the same strategy,” says Leo Poon, professor in the school of public health at Hong Kong University. His group has shared the test details — and in some cases reagents — with the WHO and with over 30 labs in Asia, Africa, the Middle-East and South America.

The PCR test is highly sensitive, if used correctly. “In our hands, we have a sensitivity of ten copies per reaction,” says Poon. However, false negatives — the failure to detect virus in infected patients — can be a significant problem in high-throughput settings operating under severe pressure. The correct operation of the test is crucial. What’s more, there are still uncertainties around the kinetics of SARS-CoV-2 viral shedding, so the timing of the test may affect the result. It is also unclear still which types of clinical specimen are optimal. One recent study from Wuhan, which evaluated the performance of a fluorescence-based RT-PCR kit distributed by the Chinese Center for Disease Control and Prevention, suggests that nasopharyngeal swabs offer greater consistency than sputum samples.

In the United States, the CDC (Centers for Disease Control and Prevention) rolled out its own PCR tests to all 50 US states as well as to 30 international locations after it obtained Emergency Use Authorization from the US Food and Drug Administration (FDA) on 4 February. But state testing labs uncovered a quality issue with one element of the three-part assay. “If you scale up fast that may happen,” says Marion Koopmans, head of viroscience at Erasmus Medical Center in Rotterdam, the Netherlands, who was involved in the development of the Berlin test.

These early lab tests buy time. They give public health authorities some diagnostic tools before commercial products and kits become available at scale (Table 1). In China, genome sequencing firm BGI Group, of Shenzhen, is already up and running, producing such commercial tests. By the end of January, it had already distributed over 50,000 test kits across China. On 5 February it opened an emergency test laboratory in Wuhan that can process 10,000 samples per day. Other companies are developing their tests more slowly, but initial rollouts are imminent. Not all are relying on primer designs and protocols specified by academic laboratories or public health authorities. “We came to the conclusion that these tests are not optimal for us, in our hands,” says Stephan Ölschläger, head of marketing at Hamburg, Germany-based Altona Diagnostics. The company has its own assay platform and is developing a two-part test targeting the E gene of lineage B betacoronaviruses and a region of the S gene specific to SARS-CoV-2.

Rather than develop bespoke PCR tests, IDbyDNA is one of a handful of companies employing metagenomic nucleic acid analysis as a routine diagnostic and surveillance tool. Its existing Explify Respiratory test, which is a laboratory-developed (or ‘home brew’) test, can identify over 900 respiratory pathogens, including viruses, bacteria, fungi and parasites, by comparing unbiased metagenomic data obtained from patient samples with a large repository of sequence data. It can already detect the SARS-CoV-2 strain. “We have now updated our data with the new coronavirus and are in the process of revalidating,” says cofounder and chief medical officer Robert Schlaberg. “The modifications are all on the data analysis side.” That is, he says, an easier process than updating a physical assay. The actual data analysis takes less than an hour; the turnaround time from receipt of sample to test result is 36 hours. Although more expensive than PCR testing, the falling costs of sequencing could help to democratize this approach. The company is selling the technology as well as its testing services. “Traditional diagnostic testing and surveillance are two separate efforts,” he says. “It’s much faster having real-time monitoring of what is in circulation.”

Andrew Rambaut, of the University of Edinburgh, in Scotland, is already doing this, tracking the outbreak by analyzing the phylogenetic relationships between different SARS-CoV-2 genomes. RNA viruses have an error-prone replication process, and the genetic variations that are introduced constitute a molecular clock that can provide insights into the initial emergence and ongoing evolution of the virus. “The molecular clock provides a way of changing the genetic changes into time,” he says. An initial analysis, based on 90 publicly available genomes, suggests that SARS-CoV-2 emerged not long before the first cases of pneumonia in Wuhan occurred. “It doesn’t predate the timing of the disease detection that much,” says Koopmans.

Epidemiologists have also adapted digital surveillance tools to track the new coronavirus outbreak by automatically monitoring structured and unstructured electronic information from official and unofficial sources. The Center for Systems Science and Engineering at Johns Hopkins University has developed a web-based dashboard to visualize and track reported cases of SARS-CoV-2 in real time. The underlying data are freely available to scientists through a GitHub repository. The dashboard’s primary source is DXY, an online resource run by members of the Chinese medical community, who aggregate official and media reports on the development of the epidemic. HealthMap.org, developed more than a decade ago to track infectious disease outbreaks, has also released an interface specific for SARS-CoV-2. Crowdsourcing and automated information retrieval have also been harnessed to develop a shared electronic line list for tracking the SARS-CoV-2 outbreak, case by case. As of 19 February, the list contained demographic, geographic and clinical information on over 7,800 individual cases, which allows researchers to conduct analyses to understand patterns of spread. “A lot of groups are using the data, so that’s one encouraging thing,” says Moritz Krämer, research fellow at the Department of Zoology, Oxford University, who set up the resource. The present crisis is, he says, characterized by a much stronger data-sharing ethos than previous epidemics. “I think we’ve learned.”

Myriad uncertainties still surround the trajectory of the epidemic. On the positive side, containment efforts outside of China appear, so far at least, to be successful. The number of confirmed cases in other countries remains low, and there is little evidence at this point of widespread community transmission. In China, however, and particularly in Hubei province — the epicenter of the outbreak, which remains in lockdown — the picture is still unclear. The majority of cases have not yet resolved. Moreover, the size of the ‘iceberg’ is undefined: it is still too early to know whether further undetected reservoirs of infection remain. There is no immediate end in sight to the extraordinary ordeal millions of people are enduring in the cities of Hubei.

Table 1 | Selected diagnostic tests for SARS-CoV-2

Developer

Test

Description

Current status

Chinese National Institute for Viral Disease Control and Prevention

New coronavirus nucleic acid assay

Primers and probes for detecting the novel coronavirus with RT-PCR

In widespread distribution in China

US Centers for Disease Control and Prevention (CDC)

CDC 2019-nCoV real-time reverse transcriptase PCR diagnostic panel

PCR test that runs on Applied Biosystems 7500 Fast Dx RT-PCR instrument with SDS 1.4 software

Emergency Use Authorization (EUA) granted by the FDA on 4 February

University of Hong Kong

Real-time reverse transcriptase PCR assays

Two single-step quantitative RT reverse transcription PCR assays for N gene and ORF1b of sarbecovirus subgenus

Reference test shipped to WHO and to over 30 labs globally

Amoy Diagnostics (Xiamen, China)

Coronavirus gene detection kit

PCR-based rapid detection kit

Seeking emergency approval from China’s National Medical Products Administration

Altona Diagnostics (Hamburg, Germany)

Real-time PCR assay

Rapid detection of coronavirus RNA from respiratory samples

In development; shipping expected end of February

BGI Group (Beijing)

Real-time fluorescent RT-PCR kit for detecting 2019-nCoV

Test results delivered in several hours

Emergency approval granted by China’s National Medical Products Administration

BGI Group

2019-nCoV nucleic acid detection kit using combinatorial probe-anchor synthesis method

Metagenomic sequencing kit for monitoring novel coronavirus mutations

Emergency approval granted by China’s National Medical Products Administration

Hong Kong University of Science and Technology

On-site rapid molecular diagnostic system based on Shenzhen Shineway Technology

Integrated microfluidic PCR test employing silicon-based micro-heater module for rapid heating and processing of test samples; delivers test results in 40 minutes

Platform has European Union CE approval

Novacyt, Primerdesign

Novel Coronavirus Strain 2019-nCoV

Runs on portable genesig16 RT-PCR instrument; delivers test results in less than two hours

CE-marked research-use-only test launched 17 February; seeking FDA EUA

Thermo Fisher Scientific

TaqMan 2019-nCoV Assay Kit

Lab PCR test that runs on Applied Biosystems 7500 RT-PCR system; identifies sequences found in initial 44 SARS-CoV-2 genomes

Research use only

Qiagen (Hilden, Germany)

QIAstat-Dx Respiratory 2019-nCoV Panel

Integrated sample prep and RT-PCR detection of 21 respiratory pathogens; samples and reagents delivered in assay cartridges and analyzed in desktop QIAstat-Dx Analyzer; results delivered in one hour

Prototype panel including COVID-19 test shipped on 11 February for clinical performance assessment in China and Europe

Biomeme

Biomeme COVID-19 Go-Strips

Integrated sample prep and RNA detection test that runs on Biomeme’s mobile handheld quantitative PCR device

Research use only

Agency for Science, Technology and Research (A*STAR), MiRXES (both Singapore)

Fortitude Kit 2.0

RT-PCR SARS-CoV-2 assay

100,000 tests produced; provisional authorization received from Health Sciences Authority, Singapore

TIB Molbiol (Berlin, Germany) also via Roche Diagnostics

SARS-CoV-2 E, RdRP or N gene CE-IVD 7 virus Respiratory Panel multiplex RT-PCR

1-step RT PCR

Launched January. 20,000 kits shipped to over 70 countries

doi: 10.1038/d41587-020-00002-2

Updates & Corrections

  • Update 21 February 2020: In the original version of this article, the test developed by Altona Diagnostics was described as targeting the F gene when it should have said the S gene. The error has been corrected.Table contents were updated on Feb 26, 2010 with information from TIB Molbiol.

Competing Financial Interests

https://www.nejm.org/doi/full/10.1056/NEJMoa030747

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