Virus World

Viral levels in people infected with SARS-CoV-2 in a specific town or city could be used to assess whether the epidemic there has passed its peak. A common test for SARS-CoV-2 allows doctors to measure an infected person’s ‘viral load’, an indicator of the amount of virus in their body. James Hay at the Harvard T.H. Chan School of Public Health in Boston, Massachusetts, and his colleagues, used modelling to show that the viral loads of a population correlate with the rate of viral spread in that population (J. A. Hay et al. Preprint at medRxiv https://doi.org/ghfm73; 2020).

Early in an epidemic, the average infected person has been recently exposed to the virus and therefore has a high viral load. Later in the epidemic, the average infected person has had the virus for longer and has a low viral load. As a result, a snapshot of the viral-load distribution in a random sample of a population can reveal whether cases in that population are on the rise or are declining, the researchers say. They add that their method is less susceptible to biases from changing COVID-testing practices than simply counting daily cases. The findings have not yet been peer reviewed.

Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The most widely used method of COVID-19 diagnostics is a reverse transcription polymerase chain reaction (RT-PCR) assay, to detect the presence of SARS-CoV-2 RNA in patient samples, typically from nasopharyngeal swabs. RNA extraction is a major bottleneck in current COVID-19 testing, in terms of turn-around, logistics, component availability and cost, which delays or completely precludes COVID-19 diagnostics in many settings.

Efforts to simplify the current methods are critical, as increased diagnostic availability and efficiency would benefit patient care and infection control. Here, we describe methods to circumvent RNA extraction in COVID-19 testing by performing RT-PCR directly on heat-inactivated subject samples as well as samples lysed with readily available detergents. Our data, including benchmarking with 597 clinically diagnosed patient samples against a standardised and sensitive diagnostic system, show that direct RT-PCR is a viable option to extraction-based COVID-19 diagnostics. Furthermore, using controlled amounts of active SARS-CoV-2, we evaluated performance of generic buffers as sample medium for the direct RT-PCR assay, identifying several suitable formulations. We also confirmed the effectiveness of heat inactivation of SARS-CoV-2 by plaque assay. Significant savings in terms of time and cost can be achieved by embracing RNA-extraction-free protocols, that feed directly into the established PCR-based testing pipeline. This could aid the expansion of COVID-19 testing.

Preprint in medRxiv (August 5, 2020):

 https://doi.org/10.1101/2020.04.17.20067348

The search for SARS-CoV-2 RNA in 60 saliva samples yielded the same results as conventional nasal swab tests taken from the same patients. There are now more options for COVID-19 testing as the US Food and Drug Administration gave emergency use authorization on April 13 for a saliva-based test, providing an alternative to the swab testing currently performed.

“You want to be in all types of situations with all types of options so that we can have as much testing as possible in whatever form is suitable,” Amesh Adalja, who works on infectious disease and pandemic preparedness at Johns Hopkins University and is not involved with the development of the test, tells the Associated Press. Currently, testing for COVID-19 involves a healthcare professional inserting a swab into each nostril, one at a time, to the nasopharynx at the back of the nasal cavity, gently scraping the tissue to collect material, and sending off for analysis, according to UC Davis Health. This method is cumbersome as it needs to be done by a qualified worker wearing fresh gloves and other personal protective equipment (PPE), which are in short supply. Additionally, many areas are experiencing a lack of tests available or a large backlog of samples to process.

The collection of a saliva sample requires spitting into a tube, resulting in a much less invasive procedure without tying up large amounts of PPE. Per the Food and Drug Administration’s (FDA) instructions, the testing would still occur in a healthcare setting under the supervision of a qualified professional....

Performance Letter by FDA:

https://www.fda.gov/media/136875/download

The Omicron variant is characterised by more than 50 distinct mutations, the majority of which are located in the spike protein. The implications of these mutations for disease transmission, tissue tropism and diagnostic testing are still to be determined. We evaluated the relative performance of saliva and mid-turbinate swabs as RT-PCR samples for the Delta and Omicron variants. The positive percent agreement (PPA) of saliva swabs and mid-turbinate swabs to a composite standard was 71% (95% CI: 53-84%) and 100% (95% CI: 89-100%), respectively, for the Delta variant. However, for the Omicron variant saliva and mid-turbinate swabs had a 100% (95% CI: 90-100%) and 86% (95% CI: 71-94%) PPA, respectively. This finding supports ex-vivo data of altered tissue tropism from other labs for the Omicron variant. Reassessment of the diagnostic testing standard-of-care may be required as the Omicron variant becomes the dominant variant worldwide.

Preprint available at medRxiv (Dec. 24, 2021):

https://doi.org/10.1101/2021.12.22.21268246 

Even as the United States ramped up coronavirus testing from about 100,000 per week in mid-March to more than 5 million per week in late July, the country fell further behind in stemming the spread of the virus. Now, diagnostics experts, public health officials, and epidemiologists are calling for a radical shift in testing strategy: away from diagnosing people who have symptoms or were exposed and toward screening whole populations using faster, cheaper, sometimes less accurate tests. By making it possible to identify and isolate infected individuals more quickly, proponents say, the shift would slow the virus' spread, key to safely reopening schools, factories, and offices. “America faces an impending disaster,” says Rajiv Shah, president of the Rockefeller Foundation. Testing, he says, needs to focus on “massively increasing availability of fast, inexpensive screening tests to identify asymptomatic Americans who carry the virus. Today, we are conducting too few of these types of tests.” Rebecca Smith, an epidemiologist at the University of Illinois, Urbana-Champaign (UIUC), agrees. To stop outbreaks from overwhelming communities, she says, “we need fast, frequent testing,” which could mean faster versions of existing RNA tests or new kinds of tests aimed at detecting viral proteins. But researchers say the federal government will need to provide major financial backing for the push.

Today, COVID-19 testing relies primarily on the polymerase chain reaction (PCR), a technique to amplify the virus' genetic material, making it easy to detect. If administered properly, such tests are highly accurate, spotting positive cases nearly 100% of the time. That accuracy is vital for decisions about treating individual patients. But PCR tests cost about $100 each, require specialized machinery and reagents, and typically take at least 1 to 2 days to return results. The recent increase in coronavirus cases across the United States has added to the delay, pushing wait times to 2 weeks in some places. While they wait, people who are infected but don't yet know it may continue to interact with others and spread the virus. And if their infective period ends before they get their results, isolating them won't help. “It's like calling the fire department after your house burns to the ground,” says A. David Paltiel, an operations research expert at the Yale School of Public Health. “You can't play catch up with this virus.” A 24 July preprint on medRxiv underscored the downsides of slow tests. Shixiong Hu, a researcher with the Hunan Provincial Center for Disease Control and Prevention, and his colleagues followed 1178 people who tested positive for SARS-CoV-2 from January to April and tested their 15,648 contacts, defined as people who had been within 1 meter of a positive person between 2 days before and 14 days after the person's symptoms began. Based on which contacts were infected and when, the researchers estimated that people were most likely to spread the virus 1.8 days before the onset of symptoms. The finding suggests testing people only when they show symptoms and giving them test results days to weeks later does little to slow viral spread, says Daniel Larremore, an applied mathematician at the University of Colorado, Boulder...

This Viewpoint discusses the 2 most common categories of testing to diagnose SARS-CoV-2—real-time PCR to identify viral RNA and serological diagnosis of IgG and IgM antibodies to assess immune response—and estimates time intervals for test positivity by specimen source to help clinician.

Thus far, the most commonly used and reliable test for diagnosis of COVID-19 has been the RT-PCR test performed using nasopharyngeal swabs or other upper respiratory tract specimens, including throat swab or, more recently, saliva. A variety of RNA gene targets are used by different manufacturers, with most tests targeting 1 or more of the envelope (env), nucleocapsid (N), spike (S), RNA-dependent RNA polymerase (RdRp), and ORF1 genes. The sensitivities of the tests to individual genes are comparable according to comparison studies except the RdRp-SARSr (Charité) primer probe, which has a slightly lower sensitivity likely due to a mismatch in the reverse primer.

In most individuals with symptomatic COVID-19 infection, viral RNA in the nasopharyngeal swab as measured by the cycle threshold (Ct) becomes detectable as early as day 1 of symptoms and peaks within the first week of symptom onset. The Ct is the number of replication cycles required to produce a fluorescent signal, with lower Ct values representing higher viral RNA loads. A Ct value less than 40 is clinically reported as PCR positive. This positivity starts to decline by week 3 and subsequently becomes undetectable. However, the Ct values obtained in severely ill hospitalized patients are lower than the Ct values of mild cases, and PCR positivity may persist beyond 3 weeks after illness onset when most mild cases will yield a negative result.However, a “positive” PCR result reflects only the detection of viral RNA and does not necessarily indicate presence of viable virus....

Published in JAMA (May 6, 2020):

https://doi.org/10.1001/jama.2020.8259