Thorough research on nine infected individuals in Germany shows important details on the natural course of SARS-CoV-2 infection, infectivity from an infected individual, and ability to detect the RNA via rRT-PCR, live virus assay, and neutralizing antibody testing
It was about five years ago, starting at a new employer SeraCare Life Sciences, that I first heard the word ‘seroconversion’. The CEO of SeraCare once explained to me the provenance of a company called Boston Biomedica Inc. back in the 1980’s, during the HIV epidemic, that was collecting leftover hospital waste (in the form of unwanted patient blood and plasma). It was noticed from serial blood draws of HIV-infected individuals the development of antibodies against the virus, and having these known-positive, seroconverted plasma samples were vital for the diagnostics industry to refine the sensitivity and specificity of the detection assays developed those 40 to 50 years ago.
The improvement was dramatic, being able drive sensitivity by several logs (this was some 10 years before the time that rRT-PCR was used routinely as a clinical diagnostics tool) and greatly assisting in the overall effort of an ongoing epidemic, both for diagnosis and to follow the course of treatment with novel therapies in affected individuals.
More about seroconversion and SARS-CoV-2 infection
You will be hearing much more about antibody testing (also known as serology testing) in the general population in the coming weeks and months as we work our way out of local lockdown orders. While valid arguments can poke holes in the Stanford methodology (here’s one Twitter thread, a second Twitter thread, and for good measure here’s a third one) overall the message is positive. There are a lot more people who have been exposed to the virus than originally thought (based upon individuals with moderate or severe COVID-19 disease), and clear consequences for highly consequential decision-making by our political leaders (whether at the local, state or national level).
It was only last night a flurry of additional information came in: first, in North Carolina, 253 out of 259 SARS-CoV-2-positive inmates had no symptoms. Then in Boston in the hard-hit area of Chelsea, Massachusetts General Hospital conducted a random sampling of individuals in the community with a serological antibody test, specifically excluding those who previously tested positive by rRT-PCR. They found that 64 / 200 (32%) tested antibody-positive against SARS-CoV-2. And then there was a report from the US Navy: the Theodore Roosevelt aircraft carrier, where the military is testing the entire crew of 4,800. (I had little idea an aircraft carrier had that many personnel in it!) While 92% complete, ‘roughly 60 percent of the over 600 sailors who tested positive so far have not shown symptoms of COVID-19’.
On top of all these, is a reminder about Diamond Princess case. The Diamond Princess was a cruise ship placed under quarantine 5 February 2020 with 3,711 passengers and crew; by 21 February there were 634 individuals testing positive for SARS-CoV-2, and a systematic analysis published in mid-March estimated a full 17.9% of those infected showed no symptoms. In addition, asymptomatic spread in Singapore has been estimated at 48% of the known SARS-CoV-2 infected cases (as a result of their testing and contact tracing effort), and in Tianjin asymptomatic spread at 62%. Here’s the MedRxiv paper if you were interested.
The consequences of asymptomatic spread, and exposure to a wider population than first anticipated, gives optimism that the Infected Fatality Ratio (IFR) actually is as low as the Stanford group estimated (0.14%) for Santa Clara County. We can expect a lot more work on this.
The virological assessment of nine cases of COVID-19 from a Munich Germany Cluster
This paper, published in Nature as an ‘accelerated article’ on 1 April 2020, examines very closely some of the first patients in Germany with COVID-19 disease, all related to this widely-publicized report in the New England Journal of Medicine of human-to-human spread during a business meeting in Munich Germany of an automobile-parts manufacturer.
The visiting employee from Shanghai had mild symptoms; the individual did not know at the time he was infected with SARS-CoV-2 when he visited Munich on 23 January 2020, although his parents lived in Wuhan and showed symptoms upon visiting Shanghai a few days before this individual traveled to Germany. Per this Lancet preprint paper, there were four people at the convertible-top manufacturer in the Munich area that were infected by the colleague from Shanghai and detected 27-29 January 2020; another 16 individuals testing positive by rRT-PCR by 19 February 2020. All the individuals infected were previously identified by contact tracing as ‘high-risk contacts’ of either the primary case or the subsequent cases.
In this Nature preprint, titled “Virological assessment of hospitalized patients with COVID-2019” by Wölfel and Wendtner et al. is remarkable for its depth, looking at the course of the disease with a variety of methods. They used rRT-PCR of various sample types: swabs (both nasopharyngeal NP and throat), sputum, and stool; they cultured live virus; they looked at seroconversion; they even assayed for presence of neutralizing antibodies. This was done from day 2 of the onset of symptoms from nine individuals through up to 28 days, when the rRT-PCR went undetectable across sample types.
Reverse transcriptase Real-Time PCR (rRT-PCR) data across sample types
A few days ago we showed here the variability in SARS-CoV-2 shedding patterns across 20 patients. This paper with nine patients reveals a similar extreme range of viral molecules, from 10^3 up to 10^8 in count (that’s five logarithms of range, or 10,000-fold differences) and individuals showing negative results as soon as day 6 after symptom onset.
Figure 1b of Wölfel and Wendtner Nature 2020, “Hallmarks of viral shedding in aggregated samples”
Presence of culturable, intact SARS-CoV-2 virus and seroconversion
It was remarkable to me to read up on how coronaviruses are cultured in a line called Vero, derived from monkey epithelial kidney cells. The paper goes on to look at live SARS-CoV-2 virus, only being able to show infectivity of the cell culture model up to day 7. Compare this to the figure above, where virus is detected in swabs up to day 20.
In addition, seroconversion was shown starting from day 5 and all nine patients seroconverted (i.e. showed IgG / IgM against SARS-CoV-2 spike protein) by day 15.
This is evidence (although the paper does make a few disclaimers) that after day 8 of onset of symptoms no live virus could be isolated, although RNA from the virus could be detected per figure 1b. It is worthwhile to note the nine individuals with COVID-19 were mild cases of disease even though they were hospitalized since they were the first cases in Germany. The authors also note in their conclusion, ‘measures to contain viral spread should aim at droplet- rather than fomite-based transmission’. (Fomites are the infective particulates that reside on a contaminated surface.)
They also state “based on the present findings, early discharge with ensuing home isolation could be chosen for patients who are beyond day 10 of symptoms with less than 100,000 viral RNA copies per mL of sputum. Both criteria predict that there is little residual risk of infectivity, based on cell culture.”
Presence of neutralizing antibodies
This paper also presented data looking at SARS-CoV-2-specific antibodies, ruling out cross-reactive antibodies against other coronaviruses. There are four endemic human coronaviruses (called HCoV-HKU1, -OC43, -NL63, and -299E), and their respective cloned spike proteins were transiently expressed in Vero B2 cells (along with SARS-CoV-2 spike protein). A test called a Plaque Reduction Neutralization Test causing plaque reductions of 90% (PRNT90) uses serial dilutions (1:10, 1:1000 etc) of patient serum samples, demonstrates the ‘blocking action’ of the serum’s antibodies to prevent live virus from infecting the Vero cells in culture, and fluorescence microscopy (example below) shows the presence of the neutralizing antibody against the specific virus’ spike protein, whether SARS-CoV-2 or one of the other HCoV variations.
In general there is a low titer of neutralizing antibodies in coronavirus infections, and thus a sensitive neutralization assay is needed which they have developed, and the authors call for a simpler ‘microneutralization test format’ for routine applications. Will be sure to be on the lookout for this development.
Part of Extended Data Figure 2 from Wölfel and Wendtner Nature 2020, “Hallmarks of viral shedding in aggregated samples”, showing recombinant SARS-CoV-2 spike-based immunofluorescence (Alexa488) demonstrating seroconversion of Patient #4
A few summary conclusions
This well-done study shows an extremely wide range of rRT-PCR quantitation from a variety of sample types, indicates virus in stool persists long after virus is cleared from the throat and sputum, seroconversion by the second week of symptoms, and presence of neutralizing antibodies. Here is one example plot from Patient #4 cropped from Figure 2 of their paper.
Figure 2 (one of nine panels) from Wölfel and Wendtner Nature 2020, “Hallmarks of viral shedding in aggregated samples”. Viral load kinetics, seroconversion and clinical observation in individual cases.
A few of the authors observations were notable: there was no co-infection with any other virus among the nine patients in this paper (rhinovirus, enterovirus, RSV, adenovirus, influenza A and B, or other coronaviruses, among several others). They authors also note ‘critically, the majority of patients in the present study seemed to be already beyond their shedding peak in upper respiratory tract samples when first tested’.
These patients were sampled at only day 2 of symptom onset, giving support to the growing evidence of asymptomatic spread.
This is a very useful “journal club” summary of an exciting preprint.
To me, Figure 2’s findings are important; I haven’t seen this type of granular picture of viral loads before. It allows us to form a mental picture of what happens in the days and weeks post-infection. Some observations:
* Along the lines of the saliva EUA discussed a few days ago here at NGT, sputum is sometimes better than a swab (patients 1, 2, 3, 4, 7, 8) and at other times non-inferior (patients 10, 14, 16). It’s never worse! If validated, this could lead to a major streamlining of test procedure.
* Upper respiratory shedding is usually higher early on (day 2-4) and declines as the infection resolves. High levels can be really high, a swab (or a ml of sputum) contained 1E7 viral genomes in 6 of 9 patients, in 3 cases, when they were coughing. Is this combination enough to qualify a person as a superspreader? Perhaps, or perhaps a larger sample would reveal a small fraction of patients who experience yet higher viral loads. I wouldn’t want to be around patient 2 on day 4 (coughing, and shedding 300,000,000 viral genomes per ml of sputum).
* To place this in the context of the Abbott ID Now PoC test: (from memory, not exact…) the test takes about 10 ul fluid, circa 1% of the load in a swab or an ml of sputum. Its Limit Of Detection is better than 1E2 (100) viral genomes. So in terms of Fig. 2, its LOD should be anything at or above 1E4. Lab-based tests that are 10x more sensitive should give a positive signal to anything at or above 1E3.
* Stool is potentially more important as a vector than has been appreciated — it often contains as much viral genomes, gram for gram, as sputum. Diarrhea could be of special concern. It’ll be important to determine if “viral genomes” translates into infectious virus particles — might or might not.
These authors wrote a great paper!