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Just as Brazil surpassed 200,000 deaths from COVID-19 on January 7, news from Bahia added another layer of concern: A platform case report in a preprint detailed the first case of reinfection in that state, apparently caused by a new strain, one having the E484K mutation.
That variant, now called Brazil P.1, has migrated to the United States. The Minnesota Department of Health announced on January 25 the nation’s first known COVID-19 case associated with it.
The mutation is located in the protein gene of the virus’ spike, which forms the crown structure of coronaviruses and is responsible for the virus’ binding to human cells. The E484K mutation is now the focus because it’s associated with mutations that escape the immune system’s neutralizing antibodies.
“This mutation is at the center of worldwide concern, and it is the first time that it has appeared in a reinfection,” the study’s first author, Bruno Solano de Freitas Souza, MD, a researcher at the Salvador regional unit of Instituto D’Or of Teaching and Research, based at Hospital São Rafael, Salvador, Brazil, explained to Medscape Medical News.
“We will wait for the sample from Bahia to confirm the case from the perspective of the Ministry of Health’s surveillance network,” said Fernando Motta, PhD, deputy head of the Laboratory for Respiratory Virus and Measles at the Oswaldo Cruz Institute, which acts as a national reference center for respiratory viruses with the Ministry of Health (MS) and as a reference for the World Health Organization.
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A Case of Reinfection
The case patient that led to the alarm was a 45-year-old woman who is a healthcare executive. She had no comorbidities. The team had been following healthcare professionals and patients who had tested positive on reverse-transcription polymerase chain reaction (RT-PCR) testing more than once to understand whether they represented cases of prolonged viral persistence or new infections.
The woman had symptoms of viral infection on two occasions (May 26 and October 26). On both occasions, results of RT-PCR testing for SARS-CoV-2 on nasopharyngeal samples were positive. In the first episode, the patient had had diarrhea, myalgia, asthenia, and odynophagia for about 7 days. She returned to activities 21 days later. In the second episode, she had more severe symptoms that lasted longer, but she still did not require hospitalization.
“It was the first confirmed case of reinfection in Bahia, and in the second episode, we observed a mutation that could have an impact on the ability of antibodies to neutralize the virus,” Souza said. “The research continues with the investigation of cases in which the patient has a positive SARS-CoV-2 RT-PCR more than once in an interval greater than 45 days, to have a higher level of evidence.”
He stressed that “it is very important to reinforce measures to control the pandemic, social distance, use of masks, and speed up vaccination to be able to control the circulation of the virus, while monitoring the evolution of it.”
On Alert for More Cases
A person who twice tests positive for SARS-CoV-2 on real-time RT-PCR is suspected of having been reinfected, provided 90 or more days have elapsed between the two episodes, regardless of the condition observed. To confirm the suspected case, the samples must be sent to reference laboratories according to a plan established by the Ministry of Health in Brazil.
“We communicated this case of reinfection to the MS in early December 2020. And the second sample already had the E484K mutation on the spike, as in the case of Bahia,” said Motta.
The first step in differentiating reinfection from persistence is to observe differences in the genotyping of the virus. For the technique to be successful, Souza said, researchers need a large amount of viral genetic material, which usually cannot be obtained.
“That is why there are many more suspected than confirmed cases,” he explained. He admitted that although there are few cases, “it is increasingly clear that reinfection is a reality.”
Markers of Mutations
What worried the researchers most was not only the possibility of reinfection but also the fact that preliminary analyses showed a specific mutation.
“The E484K mutation is present in a group of variants identified in South Africa that have been associated with increased infectivity and has been observed in a strain recently described in Brazil,” Souza said.
Mutations are expected, appear spontaneously, and in most cases have no effects on transmission or clinical outcome — they are simply used as markers and are useful for contact tracing or studying transmission routes. But some mutations can last because they provide an advantage for the pathogen, even if only momentary. In the case of SARS-CoV-2, mutations in the protein spike gene (S) are relevant because they may give clues to that advantage — as well as to changes in infectivity, transmission potential, antibodies, and response to vaccines.
A variant of the virus that has eight changes that affect the protein S gene — and several others in different genes — is behind the increase in the number of cases in London and southeastern England. Researchers from the Faculties of Medicine and Dentistry of the University of São Paulo, Ribeirão Preto campus, identified one of the factors that made this new variant — classified as B.1.1.7 — more infectious.
With bioinformatics tools, they found that the protein S gene in the new viral strain has a stronger molecular interaction with the ACE2 receptor, which is on the surface of human cells and to which the virus binds, making infection possible. The variant has already spread to the rest of the world, and the first two cases have been confirmed in Brazil by the Adolf Lutz Institute.
The alert for a new variant in Africa — similar to B.1.1.7 in the United Kingdom in that it carries nine changes in protein S at position 501 — was made by the Brazilian virologist Tulio de Oliveira, PhD.
“We found that this strain seems to be spreading much faster,” Oliveira, who is with the University of KwaZulu Natal, told the journal Science. His work first alerted British scientists to the importance of the position N501Y.
“The new variants just described in the United Kingdom and South Africa are slightly more transmissible and have already been identified in cases imported into Brazil,” Motta said. “Unfortunately, we believe it is only a matter of time before it becomes indigenous.”
The Viral Family Grows
Viruses such as SARS-CoV-2 are classified into strains on the basis of small differences in their genetic material. Since December 26, in addition to the British and South African variants, it appears the Carioca lineage also is a player.
In a preprint article, researchers analyzed the evolution of the epidemic in Rio de Janeiro from April until just before the new increase in incidence, in December. They compared the complete sequences of the viral genome of 180 patients from different municipalities. The study, which is being jointly conducted by members of the Federal University of Rio de Janeiro (UFRJ) and the National Laboratory for Scientific Computing, identified a new variant of SARS-CoV-2 that has five unique mutations (from one of the predominant strains). Concern arose because, in addition to those five genetic changes, many of the samples had a sixth — the well-known E484K mutation.
“The three lines — the UK, South Africa, and Brazil — were almost synchronous publications, but there is no clear evidence that they have any kind of common ancestry,” Carolina M. Voloch, PhD, the article’s first author and a biologist and researcher at the Molecular Virology Laboratory and associate professor in the Department of Genetics at UFRJ, told Medscape Medical News.
Voloch’s research focuses on the use of bioinformatics tools to study the molecular, phylogenetic, and genomic evolution of viruses.
“The emergence of new strains is common for viruses,” she said. “It can be happening anywhere in the world at any time.”
She stressed that identifying when mutations emerge will help to define the new Brazilian lineage. Researchers are working to determine whether the neutralizing antibodies of patients who have been infected with other strains respond to this Rio de Janeiro strain.
“We hope to soon be sharing these results,” Voloch said.
The article’s authors estimate that the new strain likely appeared in early July. They say more analysis is needed to predict whether the changes have a major effect on viral infectivity, the host’s immune response, or the severity of the disease. Asked about the lineage that caused the reinfection in Bahia, Voloch said she hadn’t yet contacted the authors to conduct a joint analysis but said the data disclosed in the preprint would not represent the same variant.
“There are only two of the five mutations that characterize the Rio de Janeiro lineage. However, it has the E484K mutation that is present in more than 94% of the samples of the new variant of Rio,” she said.
She added that there’s a possibility of reinfection by the lineage that’s circulating in Rio de Janeiro and in other states, as well as countries such as the United States, the United Kingdom, and Japan.
“The carioca virus is being exported to the rest of the world,” Voloch said.
Virus’ Diversity Still Unknown
Researchers now know that SARS-CoV-2 probably circulated silently in Brazil as early as February 2020 and reached all the nation’s regions before air travel was restricted. Since the first half of 2020, there have been two predominant strains.
“More than a dozen strains have been identified in Brazil, but more important than counting strains to identify the speed with which they arise — which is directly associated with the rate of infection, which is very high in the country,” said Motta.
The so-called variant of Rio de Janeiro, he said, has also been detected in other states in four regions of Brazil. The key to documenting variants is to get a more representative sample with genomes from other parts of the country.
As of January 10, a total of 347,000 complete genome sequences had been shared globally through open databases since SARS-CoV-2 was first identified, but the contribution of countries is uneven. Although the cost and complexity of genetic sequencing has dropped significantly over time, effective sequencing programs still require substantial investments in personnel, equipment, reagents, and bioinformatics infrastructure.
According to Voloch, it will only be possible to combat the new coronavirus by knowing its diversity and understanding how it evolves. The Fiocruz Genomic Network has made an infographic available so researchers can track the strains circulating in Brazil. It’s the result of collaboration between researchers from Fiocruz and the GISAID Initiative, an international partnership that promotes rapid data sharing.
As of January 5, researchers in Brazil had studied 1897 genomes — not nearly enough, scientists said.
“In Brazil, there is little testing and even less sequencing,” lamented Souza.
“In the UK, one in 600 cases is sequenced. In Brazil it is less than one in 10 million cases,” Voloch added.
So far, no decisive factors for public health, such as greater virulence or greater transmissibility, have been identified in any of the strains established in Brazil. The million-dollar question is whether the emergence of new strains could have an impact on the effectiveness of vaccines being administered today.
“In one way or another, the vaccine is our best bet ever, even if in the future we identify escapist mutants and have to modify it,” Motta said. “It is what we do annually with influenza.”
This article was originally translated from the Portuguese edition of Medscape.
Voloch, Motta, and Souza have disclosed no relevant financial relationships.
Follow Roxana Tabakman on Twitter @roxanatabakman.