Serological test for more precise diagnosis of zika comes to market

Product developed with FAPESP’s support has been licensed by Brazil’s public health surveillance agency after trials involving more than 3,000 women. The method can detect old infections, even in patients who have had dengue or yellow fever.

By Maria Fernanda Ziegler  |  Agência FAPESP – A novel serological test to detect antibodies against zika virus in blood samples has been approved for sale in Brazil. The low-cost test is an advance on those hitherto available commercially because it can tell clinicians whether the individual is infected even after the acute phase of the disease is over and is accurate even when the patient has previously had dengue or yellow fever.

The method has been validated in trials involving more than 3,000 women in different parts of Brazil. It was developed by AdvaGen Biotech in collaboration with researchers at the University of São Paulo’s Biomedical Sciences Institute (ICB-USP) and Butantan Institute in São Paulo. The project was supported by FAPESP through the PIPE-PAPPE Grant Program, a partnership between FINEP, the Brazilian government’s innovation agency, and FAPESP via its Innovative Research in Small Business program (PIPE).

AdvaGen, which holds the patent, is based in Itu, São Paulo State, and is equipped to produce 40,000 tests per day. Having won approval from ANVISA, Brazil’s national public health surveillance agency, to market the test, the company is now validating it with the Central Public Health Laboratory (LACEN) in Brasília for use by the Stork Network antenatal program run by SUS, the national health service.

The test has also been offered to four private laboratories in Brazil and is being validated in Argentina and Colombia.

The company’s goal is to have it included in the list of antenatal tests approved by the Health Ministry and local health authorities in Brazil’s 27 states. In addition to showing whether an individual has been exposed to zika virus, it is also designed to identify cases of women who were infected during pregnancy and gave birth to babies without microcephaly. These children may have developmental complications, such as cognitive impairment and motor disability.

“Our focus is mainly on pregnant women. The test identifies whether a subject has been made immune [infected at any time], even if they’ve had dengue or yellow fever. Pregnant women who take the test and determine they’ve never been infected will take appropriate precautions, such as using insect repellent and avoiding high-risk areas. If the test detects infection during pregnancy, the subject can be monitored for a longer period, even if the baby is born without microcephaly,” said Edison Luiz Durigon, Professor of Virology at ICB-USP and one of the scientists who developed the test.

For Durigon, the test could be a strategic element in the formulation of public policy, since babies exposed to the virus during pregnancy may be born with minor brain damage that is not initially detectable but can lead in the future to cognitive impairment and other complications.

“Microcephaly is just the tip of the iceberg,” he said. “The disease is often asymptomatic. Even now, we don’t know the extent of the epidemic for lack of data. We believe that some 90% of pregnant women who have zika don’t report the disease because they have no symptoms and are unaware they’ve been infected. So many of the children born without microcephaly may suffer from dysfunctions that will be noticed only when they reach school age.”

The test identifies these specific cases, he explained, although they will then require more sophisticated exams to detect brain damage, such as CAT scans and MRI scans.

“Today, we have around 3,800 children institutionalized because of zika virus. This is a large number and refers only to children with microcephaly. How many have been affected altogether we don’t know, as there’s a shadowy area in this epidemic that we can’t see clearly. The danger is that new developments will be detected in the years ahead, such as an increase in the numbers with learning difficulties at school. This could be a consequence, not as severe as microcephaly, of course, but also very serious,” he said.

Inexpensive and highly specific

The main advantage of the test compared with those currently available is that it measures specific antibodies and can therefore identify zika in serum even from people who have had contact with closely related pathogens, such as dengue virus.

“The first outbreak of the disease in Brazil occurred in December 2015, and three serological tests were already commercially available by July 2016. However, they aren’t highly specific and can produce false positives if subjects have had dengue fever or another disease caused by a pathogen belonging to the same flavivirus genus. This is very common in several parts of Brazil where dengue is endemic,” said Danielle Bruna Leal de Oliveira, a researcher at ICB-USP’s Clinical & Molecular Virology Laboratory and principal investigator for the project.

The team of researchers developed the serological test to detect the viral protein to which immunoglobulin G (IgG) antibodies adhere during infection, thereby showing whether the subject is immune. IgG antibodies remain in the organism after infection, providing lifelong immunity.

The main difficulty associated with the method is that the viral protein in question, NS1, is very similar in all flaviviruses, including dengue, zika and yellow fever, among others. To solve the problem, the researchers at ICB-USP used an edited version of the protein, selecting only the zika-specific part of the molecule.

“It was very important to make sure there were no cross-reactions with the four types of dengue virus in subjects who had had dengue fever. That’s why we conducted trials with more than 3,000 patients to validate the product. We selected population samples in areas where dengue is endemic, such as São Paulo, Bahia and Goiás, among other states,” Durigon said.

The test is based on the methodology known as ELISA, an acronym for enzyme-linked immunosorbent assay, because it is inexpensive and affordable for most people. The platform consists of a microtiter plate with 96 tiny wells to which a viral protein recognized by the human immune system adheres. The wells are filled with blood serum from up to 94 patients, with two or more being reserved as controls. In the case of prior contact with zika, IgG antibodies adhere to the viral protein, which is later detected by colorimetric assay (positive and negative samples acquire different colorings).

According to Durigon, it is possible to tell from the number of antibodies whether the infection is recent or old. “This isn’t the point of the test, but it shows whether the subject has a lot of antibodies. If not, then the infection is likely to have occurred a year earlier, for example,” he said.

“The antibodies don’t disappear from the organism completely, but they tend to fade out over time. The good news is that even a subject with a low level of antibodies [hence, an old infection] rapidly builds up immunity again on coming into contact with the virus a second time. The immune system is highly reliable.”