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The man’s condition was already dire when he arrived at the hospital in Belgrade, Serbia. He was coughing, short of breath, and had a high fever of 39.2°C. Doctors and nurses quickly tested the oxygen levels in his blood – they were way down. Although they intubated the man, hooking his lungs up to a mechanical ventilator, he died just a few hours later.
It is a sequence of events that, sadly, has unfolded time and time again throughout the pandemic. Except that this man died on February 5, 2020, before any Covid-19 deaths were officially confirmed in Europe. Before the name “Covid-19” even existed. Back then, the man’s doctors had no idea what had killed him. They put the fatality down to pneumonia of an unknown cause.
At the time, scientists were aware that a new coronavirus had emerged in China and was spreading to Europe via a handful of overseas travellers. The man in Belgrade, however, had no recent travel history. He was aged just 56 and did not have any chronic health conditions. Now, researchers in Serbia have published a paper detailing how they found evidence of what was later named Sars-CoV-2, the virus that causes Covid-19, in tissue samples taken while the man was in hospital.
“Without any doubt, Covid-19 was the cause of death of this patient,” says co-author Aleksandra Barać, of the Clinical Centre of Serbia.
As spring 2020 rolled on and the pandemic raged across Europe, it was the flood of new cases that became the focus of scientific inquiry – not the first trickle of early ones. But during the ensuing months, researchers in the UK, Italy, France and other countries have occasionally had the opportunity to go back and test old tissue samples or re-examine case histories.
In some instances, as in Serbia, these investigators have uncovered previously undiagnosed Covid-19 infections, including deaths now known to be associated with the disease, revealing that the virus was having a greater impact in Europe than anyone realised back in January and February 2020.
Retrospective analysis is not always possible. Doctors don’t always take tissue samples from patients during their stay and, even when they do, such samples may not be retained for longer than a few days or weeks. But on those occasions when specimens have been stored long-term, allowing subsequent analysis months later, scientists have been able to provide closure to a handful of families left questioning whether their loved ones died of Covid-19 or not.
More broadly, identifying such cases helps us sharpen our blurry view of the pandemic’s frightening early days, potentially offering insights that could help us fight the next major outbreak of a new disease when – not if – it arrives.
It was the jelly-like vitreous humour from the Serbian patient’s eyeball that bore the secret of what happened to him. According to Barać, when a patient in Serbia dies within 24 hours of hospital admission, it is standard practice to perform a clinical autopsy and retain various samples, such as blood and urine, for further tests.
The man’s vitreous humour was extracted the day after he died and frozen at -20°C. Three months later, just after the Covid-19 outbreak in Europe had passed its April peak, staff at the University of Belgrade’s medical faculty recalled the man’s case and wondered whether there could be evidence in his samples that he had caught Covid-19.
They turned to the hospital’s freezers and retrieved the vitreous humour, which they then thawed. In order to find out whether Sars-CoV-2 was present in the specimen, they performed two genetic tests on the material, including a PCR analysis. Each test looked for different genetic sequences known to be contained within the virus. By doing two different tests, they practically eliminated the chance of a false positive result.
“Having that confirmatory assay is really good,” says Jeremy Rossman, a virologist at the University of Kent who was not involved in the work. “That’s giving really good assurance.”
Barać and her colleagues argue that, since the viral RNA clearly survived well, vitreous humour samples could be a key resource for other researchers looking for evidence of Covid-19 months after a person has passed away, in cases where such samples were retained.
Only rarely do hospitals keep specimens like this for extended periods, though. An NHS document available online states that, in the UK, when solid tissue samples are obtained from patients, they are kept for four to six weeks before being destroyed. Samples may not be taken at all, except via a biopsy while the patient was alive, for instance, or after death, in situations where the cause of death is unclear.
A spokesperson for Public Health England (PHE) says that it is “aware of instances where retrospective testing has identified confirmed cases, which were undetected at the time of their illness in February and March 2020. This includes one person who sadly died.” The spokesperson was unable to clarify how many previously undetected cases had been identified via retrospective analysis.
However, it recently emerged that the earliest Covid-19 death known to have occurred in the UK was uncovered using these methods. By late January 2020, 84-year-old Peter Attwood of Kent had spent weeks suffering from a pneumonia-like illness that left his doctors puzzled. He deteriorated further during a stay in hospital and eventually passed away on January 30, 2020 – a day before the first two Covid-19 cases were officially confirmed in the UK. Attwood had never left Britain in his life and therefore had no travel history that would link him to outbreaks in China or continental Europe. His doctors concluded that he had had bronchial pneumonia and secondary heart failure.
But his daughter, Jane Buckland, was unconvinced. When she heard about the sort of symptoms caused by Covid-19, she recognised them and requested that her father’s tissue samples be examined for evidence of Sars-CoV-2. Seven months later, she received a letter stating that pathologists had carried out this analysis, leading them to confirm that he had indeed had the virus. The pathologists had found viral RNA in Attwood’s lung tissue, which had been retained following his death.
“It was a shock in a way to see it,” Buckland told Sky News, adding, “I was almost positive that that was it. Myself, as a daughter. I just knew it.”
There are others, besides Buckland, who have questioned whether their loved one died because of Covid-19 at a time when there was little or no testing for the disease. One woman from Scotland who asked to remain anonymous says she strongly believes that her father, admitted to hospital in December 2019, had Covid-19. He died on January 14, 2020.
Without biological proof, it’s impossible to say for sure, notes Rossman, because Covid-19 symptoms can be so like those of other diseases. That is why confirmatory genetic analysis can be so impactful in cases where stored tissue samples make it possible to reassess what happened to a particular patient.But how probable is it that many more people were succumbing to the virus than we realised at the time? The numbers may not be in their thousands but there were almost certainly more active cases in the first weeks of 2020 than was thought at the time, says Mark Graham, a researcher at King’s College London.
“I wouldn’t be surprised that there were many more than just one or two cases circulating in January ,” he says. He and colleagues behind the Zoe symptom tracking app, which was launched in the UK in March last year, say their data shows a huge spike of symptoms associated with Covid-19 in April last year.
Logically, working backwards, the virus must have been spreading in the weeks before this surge but there are very few ways to gauge the extent of this. Modelling is one such way.
In May last year, Mark Jit at the London School of Hygiene and Tropical Medicine and colleagues published a paper that described how they used data on the number of Covid-19 patients admitted to critical care in spring 2020 to estimate the number of infections that were likely circulating in the early days of the pandemic in the UK.
“We estimated a handful of new cases every day until the last week of February, when it jumped to hundreds of cases every day, and then to thousands by early March,” he says.
Though still not certain, this is the probable backdrop to the handful of retrospectively confirmed Covid-19 cases and deaths that have emerged since. Where models paint a useful but vague picture, analysis of tissue samples has been able to zero in on individual cases, revealing the stories of people who caught the virus and survived, or did not.
This kind of analysis hasn’t just been carried out in the UK and Serbia. In France, pathologists at a hospital in the north of Paris reanalysed samples from 24 patients who tested negative for flu in December 2019 and January 2020. A sample from one of these patients, who was hospitalised that December and later recovered, was found to contain SARS-CoV-2. Doctors had retrieved the specimen from the patient nearly a month before the first official Covid-19 diagnosis in France. The pathologists who followed up only realised that it contained Sars-CoV-2 months later.
In Italy, an even earlier case has surfaced. Researchers returned to throat swab samples obtained from 39 different patients between September 2019 and February 2020. One, from a four-year-old boy with no travel history who lived near Milan, tested positive for Sars-CoV-2. The sample was originally taken on December 5 but the boy’s illness had first emerged the previous month, November, initially with coughing and a runny nose, then respiratory symptoms and vomiting a week later. The case suggests that community transmission may have been occurring in Italy roughly three months before the country confirmed its first case of Covid-19.
To grasp just how rare it is to find previously undetected evidence for SARS-CoV-2 in old tissue samples, take the study of specimens from more than 300 patients in Basel, Switzerland, published in January. A team of pathologists found evidence for an increase in bronchopneumonia between October 2019 and February 2020 in the samples, which they suggest was likely seasonal. However, they found zero evidence that any of these patients had Covid-19.
The World Health Organisation says it is monitoring such research. Rossman says that if the pandemic is brought under greater control in the coming months with the aid of vaccines then further retrospective analyses may emerge, perhaps in cases where other stored tissue samples have lain unexamined. “These events are fortunately extremely rare,” says an NHS spokesperson. “The NHS is currently undertaking its own review of these incidents as hospitals rightly continue to prioritise patient safety.”
Ultimately, improved data on early cases not only helps bring closure to families affected by the pandemic, it also helps scientists to reassess their view of when the pandemic really began. Rossman argues that, while this information may not change much about how we deal with the ongoing spread of Covid-19 in the present, having a better understanding of how the virus first took hold in those early months can still be of use in the future. “The next time something happens, we can respond better and faster,” he says.
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