By Maria Fernanda Ziegler | Agência FAPESP – Researchers in the state of São Paulo, Brazil, are developing low-cost rapid tests to diagnose COVID-19. The initiatives use different strategies to detect the virus or antibodies produced by an infected person to combat the disease. They aim to identify both infected people and those who have had the disease and may be immune, with or without symptoms.
Some of these initiatives to develop rapid tests to detect SARS-CoV-2 are offshoots from previous research to detect Zika, dengue and other viruses. “We must bear in mind that the epidemic is expected to last a fairly long time and there may be more waves of infection, so it’s essential to have different types of tests that yield epidemiological data while also planning measures that involve both social isolation and relaxation of confinement to enable people to go to work. If we increase testing, we can identify moderate and severe cases, people who have recovered, and people who are unaware they’ve been infected,” said Edison Durigon, a professor in the University of São Paulo’s Institute of Biomedical Sciences (ICB-USP) who has led several research projects relating to the coronavirus.
Researchers at ICB-USP are developing a strip test (similar to a pregnancy test) to detect the coronavirus in nasal or throat secretions in 15 minutes. The study was approved by FAPESP in the recent call for research proposals titled “Fast track supplements for projects against COVID-19”.
The research is at an advanced stage, and test kits are set to be ready in May. All the necessary inputs, such as viral antigens and antibodies, are being developed in ICB-USP’s own laboratory, avoiding the need to import reagents at a time of extremely strong demand worldwide.
“We were able to isolate the virus from patient number 1 in Brazil, and our lab produced three surface proteins that enable the pathogen to be identified in tests of this kind,” said Durigon, one of the researchers leading the project. “The study is now at the stage of inoculating the proteins into lab animals – rats, rabbits and goats – to have enough serum to produce the first rapid tests.”
According to Durigon, the COVID-19 test is being developed along the same lines in terms of research and methodology as those implemented during the Zika epidemic. “Development of this test is based on all the knowledge we acquired in previous research on Zika, namely, we isolated the virus, produced the protein and developed the test,” he said.
The proteins produced in Durigon’s laboratory can be used in the future to develop tests that detect antibodies to determine whether a person’s immune system has reacted to infection by the virus, he added. “However, at this point in the epidemic, we would rather focus on a test that diagnoses infection in 15 minutes. With this method, it isn’t necessary to wait for people to produce antibodies because we can identify the virus while it’s circulating,” he said.
On the smartphone screen
At the Center for Research and Development of Functional Materials (CDMF), a Research, Innovation and Dissemination Center (RIDC) supported by FAPESP and hosted by the Federal University of São Carlos (UFSCar), an electrochemical sensor is being developed to detect SARS-CoV-2 in the first few days after infection.
The sensor consists of zinc oxide nanoparticles capable of detecting the virus’s recombinant protein in nose and throat secretions. “This strategy enables us to identify the virus in circulation right at the start of infection, which is important to control the pandemic. It’s a rapid test that can detect initial cases and asymptomatic cases,” said Talita Mazon, a researcher at the Renato Archer Center for Information Technology in Campinas and principal investigator for the project.
According to Mazon, the test device contains a printed circuit board (like most electronic appliances) and zinc oxide nanoparticles to immobilize antibodies against the coronavirus spike proteins S1 and S2.
“We’re adapting the method we developed for Zika and dengue for use in detecting the SARS-CoV-2 recombinant protein [produced in the laboratory] via antibodies immobilized on the nanostructures,” Mazon told Agência FAPESP. “At the same time, we’re partnering with a group of researchers at the University of Campinas [UNICAMP], who have isolated the virus in the lab, to modify the sensor to the shape and size of this virus so that it can be detected directly. This means that the rapid test will be 100% local technology and we won’t need to wait for imported antibodies.”
The virus is detected by a portable device in 15 minutes using a drop of mucus from the patient’s throat. The result is shown in screen graphics displayed by a smartphone app. “We’re starting to talk to manufacturers about large-scale production,” Mazon said. “If all goes well, I expect the sensor to be widely available in about three months.”
A project underway at São Paulo State University (UNESP) in Botucatu uses a strategy based on gold nanoparticles to identify antibodies in saliva or other secretions in 15 minutes.
“We’re developing the same type of rapid test as the one imported recently by the Brazilian Health Ministry. We have experience with this type of test for other diseases, such as dengue, canine distemper, and more recently porcine circovirus. We’re adapting the methodology for use in testing for SARS-CoV-2,” said João Pessoa Araújo Júnior, a researcher at UNESP’s Botucatu Institute of Biosciences (IBB).
To develop the test, the group had to import immunoglobulin G (IgG) and immunoglobulin M (IgM), antibodies produced by defense cells to combat infection. The method consists of a chromatographic test strip containing gold nanoparticles coated with antibodies specific against SARS-CoV-2. When the strip comes into contact with patient secretions, any IgG or IgM in them will bind to the antibodies, and the color of the strip will change.
The cost of the nanostructures is low. The dengue test kit developed by the same research group costs about a dollar, although in that case, there was no need to import the antibodies.
“This is a three-in-one test: it shows whether the patient hasn’t been infected, is infected now or has been in the past. This is because IgM antibodies are produced during the acute stage of the infection, while IgG antibodies appear after the individual has theoretically become immune,” said Valber Pedrosa, a researcher at IBB-UNESP.
Rapid tests can give scientists an estimate of the actual scale of the epidemic, he added. “They’re important because in Brazil, we’re only counting hospitalized patients who test positive, but it’s also necessary to know the numbers of asymptomatic cases and immunized people as a basis for planning when to end the lockdown and how to deal with possible new waves of the disease,” Pedrosa said.
Tests in clinics/hospitals
The ICB-USP group is developing another test that gives a result in four hours and is purpose-built to be performed in hospitals or clinical analysis laboratories. It uses the serological method ELISA (enzyme-linked immunosorbent assay) to detect IgG and IgM. The main advantage is that sophisticated equipment is not required.
“ELISA can show whether a person is infected, already has antibodies or was infected at some point in the past,” Durigon said. “This test will be very useful in future stages of the epidemic because it can identify people unaware they have had been infected.”
In addition to the COVID-19 rapid tests and ELISA test now being developed, there is also the RT-PCR (real-time polymerase chain reaction) method recommended by the World Health Organization (WHO) and based on detection of the circulating virus’s RNA in infected individuals. Considered the gold standard for COVID-19 testing because of its accuracy, this method produces a result in approximately 12 hours. In Brazil, it has been used only to diagnose critical patients.
In the state of São Paulo, COVID-19 diagnostic testing is being optimized by a network of some 20 laboratories, mostly public and associated with universities or research institutions (read more at: agencia.fapesp.br/32997/).