Instituto D'Or Lab in Botafogo, Rio de Janeiro / María Tapia Seoane.

Zika, the black hole of diagnosis

By ANTONIO VILLARREAL. 25th APRIL 2016.

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RIO DE JANEIRO, Brazil — They called it the Camaçari syndrome. In this city, northeast of Brazil, surfaced the epidemic that, on March 26, 2015 - when Antonio Bandeira came to take samples of patients presenting signs of fever, conjunctivitis, joint pains and rashes - was still classified as “mysterious”.

Bandeira, a doctor at Hospital Aliança located in the nearby Salvador de Bahía, sent the blood of 24 patients to virologists Gúbio Campos and Silvia Sardi, from Universidad Federal de Bahía. First, they extracted the virus genetic material from the solution, and subjected that RNA to a process of polymerase chain reaction preceded by a reverse transcription, or what is the same, they amplified in great detail the virus genes in order to identify whether it was dengue, chikungunya, West Nile fever, Mayaro virus or whether, as eventually it turned out, Zika had reached South America.

It took them 20 days to identify it. A year later, scientists around the country are working against the clock to make the same diagnosis in 20 minutes or less.

At the Hospital Universitario Clementino Fraga Filho, in Rio de Janeiro, Izaias Loureiro, who the nurses call Passarinho - little bird in Portuguese - returned a few days ago to his post as refrigeration technician after overcoming Zika. “I came right here to see the doctor, who prescribed Tylenol 750”, an analgesic with paracetamol, “and recommended drinking plenty of water and lots of juice.”

“They suspected it was Zika, but it was never confirmed” says Loureiro. His mobile phone begins literally to chirp and Passarinho, after postponing the call, takes the opportunity to show pictures of his torso covered by the rash. “I'm ridiculous without a shirt”, he jokes.

He has not been the only worker affected in the hospital, which receives a trickle of about three new cases per day according to estimates by the infectious disease specialist, Alberto Chebabo. “Especially in the last three months the cases have increased, although it is difficult to be precise with the numbers.”

Some of the scientists and physicians interviewed in this article. From left to right, Alberto Chebabo, Antonio Gomes, Claudio Struchiner, Izaias Loureiro, Paolo Zanotto and Stevens Rehen / María Tapia Seoane.

For a primary care physician, an accurate diagnosis is complicated. Chebabo’s uses the same parameters all primary care physicians in the country use: “If they come with weakness, rash and red eyes it is usually Zika, if they present a lot of joint pain and high fever, it is rather chikungunya, dengue usually causes generalized muscle pain and headache. These are the classic symptoms of the three diseases, but we can end up confusing them.”

In the Camaçari study, which involved the discovery of Zika, authors warned that the confluence of dengue, chikungunya and Zika cases in Brazil will make the clinical or epidemiological diagnosis unreliable. These issues show the need to confirm these arboviral infections in the laboratory.

So far, diagnostic confirmation in the laboratory is restricted to pregnant women with a Zika-like clinical picture. For other patients: paracetamol, lots of juice and a suspicion.

The mysterious disease

The quick diagnose of the disease is not only key to avoiding high-risk pregnancies and that many children are born with neurological damage. It also serves to understand the disease, how does it expands and how to prevent it.

This Zika strain, from French Polynesia, seems to have found a particularly comfortable accommodation in the South American Aedes aegypti mosquitoes. Scientists are surprised by the speed with which it spreads and are intrigued by the fact that some symptoms affect more some areas than others. For example, 77% of mothers who had children with microcephaly were poor.

“I'm surprised by the speed at which the virus is moving”, says Claudio Struchiner, an epidemiologist at the Fundação Oswaldo Cruz, “but we do not know exactly how fast it is moving because the diagnosis is not done properly, we don’t have the tools to detect the infection and this is a problem.”

“We do not know exactly how fast it is moving because the diagnosis is not done properly”

The Brazilian Ministry of Health publishes the number of new cases each month. Although the most serious estimates speak of more than 1, 5 million patients infected in the country, the fact is that in the most remote areas of Brazil Zika incidence is still unknown.

In addition, according to Struchiner, “it is not clear that this epidemic has the pattern of a vector-borne disease, it does not present the same signs as dengue; people do not describe different symptoms of dengue in different areas of the country, if you have more microcephaly cases in the northeast than in other regions, there is something different there”.

The geneticist Amilcar Tanuri and his team at Universidad Federal de Rio de Janeiro were the first to sequence the Brazilian Zika genome, discovering that it was the same strain that caused an epidemic in New Caledonia in 2013. There is something in Zika that puzzles him. In some cases, people who have already overcome the virus suffer symptoms again a month later, and Zika can been detected in their urine for weeks.

“This behavior is very strange, such a virus does not usually become chronic”, reflects Tanuri. “Either it causes a disease or it is eliminated, there are exceptions in DNA viruses such as herpes or HIV, which remain years and years, but we have never seen an RNA virus remaining in the body for a long time.”

Brazilian states with Zika cases

Without Zika cases

Bogotá

Colombia

Peru

BRAZIL

Recife

Lima

Salvador

BOLIVIA

Brasilia

La Paz

Sao Paulo

pARAGUAY

Rio de Janeiro

ARGENTINA

URUGUAY

Santiago

Buenos Aires

Brazilian states with Zika cases

Without Zika cases

Bogotá

Peru

BRAZIL

Recife

Lima

Salvador

Brasilia

La Paz

BOLIVIA

Sao Paulo

Rio de Janeiro

ARGENTINA

Buenos Aires

Santiago

Brazilian states

With Zika cases

Without Zika cases

BRAZIL

Recife

Salvador

Brasilia

La Paz

BOLIVIA

Rio de Janeiro

Sao Paulo

ARGENTINA

Buenos Aires

Santiago

Boletím Epidemiológico (Ministério da Saúde do Brasil) / Luis Sevillano.

Zika's link with microcephaly also came as a surprise to the authorities, which try to adjust on the fly their clinical screening procedures to adapt themselves to the new and mysterious reality. For example, the head circumference under which a possible risk of neurological damage is suspected was previously 32 centimeters, but the ministry has lowered it half a centimeter to leave nothing to chance. “The virus reduces head circumference, although in some cases it seems to come back to normal it's just water surrounding the brain”, explains Tanuri. “Some girls were born with a normal perimeter but had abnormalities in the thalamus, cerebellum or corpus callosum.”

At Instituto D'Or de Botafogo, the neuroscientist Stevens Kastrup Rehen - considered one of the most influential researchers in the country - extracts stem cells from the skin and reprograms them in neuronal cells, with which he builds organoids that functionally mimic a brain of a few weeks old. Then, the researcher infects those mini-brains with Zika to study the damage the virus does to the cells.

Researcher observes neuron organoids or minibrains in a glass / María Tapia Seoane.

“We have observed that the infection generates apoptosis and reduces the size of the organoids after 11 days”, says Rehen. He and other researchers argue that microcephaly is just one of many other consequences of the infection, framed in what could well be a syndrome.

Tanuri, one of the authors of a study that tries to classify this syndrome - to be published soon in the New England Journal of Medicine - lists other injuries caused by the virus, “involution of the uterus, calcifications in the brain, increased ventricle, skeletal abnormalities, arthrogryposis... in my opinion it is a congenital syndrome”.

At the same time, Rehen is trying to help the mini-brains with different drugs already available on the market that can be used immediately to minimize the damage caused by the infection. “We already have some candidates and hope to announce results in one or two months”, he says.

The difficulty of diagnosis

Foreign companies - the first one was the German Genekam - have developed and exported to Brazil different versions of Zika detection tests. “The problem is that they are not validated”, laments Tanuri, “they work properly in their hands, but have not been tested in Brazilian patients already infected by many other arbovirus”. On the other hand, “the tests we produce here are handcrafted, and cannot be implemented on a wide scale.”

Since it was discovered in rhesus macaques in Uganda in 1946 - to travel afterwards to Micronesia or Easter Island - this virus had never behaved like that. It was considered a mild form of dengue, a temporal fever. This partly explains that, in 2015, the search for a diagnostic test against Zika had to start from scratch.

In his office at the Butantán Institute in Sao Paulo, Jorge Kalil, a veteran immunologist and director of the center, recalls that “when Zika appeared in Polynesia, the Pasteur Institute performed a diagnostic test for the presence of immunoglobulin using a protein fragment of the virus.”

Since it was discovered in rhesus macaques in Uganda in 1946 this virus had never behaved like that

Kalil is one of those involved in the development of a new, fast, reliable and specific serologic test for Zika. “The reliable diagnostic methods we have today detect virus through gene amplification by PCR, and are very useful, but the big limitation we find is that they detect the virus only when it is present, if a pregnant woman had the virus weeks before it does not appear, but the fetus can suffer from brain damage”, he says. “The virus is able to avoid the immune system and continue to circulate in the body, it disappears from the blood but remains in saliva, semen, urine...”

Apart from this problem, there is also the complexity of the immune response. As Struchiner states, “there are different dengue serotypes and you cannot know which one has infected you in the early stages of the disease because the test is not that specific, and with Zika we find the same problem, we lack tools which allow us to observe infection markers similar to Zika.”

Recently, in a non-structural protein of the Zika virus, researchers identified a region that has not homology with dengue, with a great immunogenicity - subjects developed many antibodies against it. Fragment 3 of E-protein emerged then as a Rosetta Stone, since it allows the virus detention even months later, what Kalil defined as an “immune scar.” The trail of Zika.

Olympics, politics and mosquitoes

If a single infected person was able to cause an epidemic that has spread to virtually all countries between Paraguay and Mexico, what can happen when tens of thousands of people around the world come to the Olympic Games in Rio, the world's largest sporting event of the year?

“There are many discussions about the Olympics”, says Tanuri, whose group is in talks with the International Olympic Committee to set up a laboratory in the Olympic Village where the athletes can be tested for the virus, “we could have results in 24 or 48 hours” says the geneticist. “I think the athletes will be highly protected, the problem is the people, the suburbs of Rio... it is a calamity.”

An Emergency Unit in downtown Rio de Janeiro / María Tapia Seoane.

In the city, posters with recommendations about Zika exceed by several orders of magnitude the Olympic reminders. Next 5 August, the Olympics curtain will rise, and this pressure also affects Brazilian researchers, who face an announced budget cut of 50%. For many of them, this race to develop a diagnostic test, map the Zika virus and ultimately save the country from the disease in the eyes of the world is an opportunity to receive additional funding, but also to claim the role of science.

A few kilometers from Butantán, in a spacious hall decorated with enlarged microscope samples, framed and tinted in different colors, Paolo Zanotto, a microbiologist at the Instituto de Investigación Biomédica de la Universidad de Sao Paulo, is about to attend a press conference. In one hour, Zanotto will announce to the country that the serological diagnostic test for Zika has been validated.

On his way to an ethics committee meeting of the university, which takes place in the room next door, a lady approaches Zanotto and whispers “habemus minister.” She refers to the former president Lula da Silva, who was appointed by President Dilma Rousseff that 16 March.

The validation of the test means that, finally, an antigen that reacts to the scar left by Zika has been found. Now they can start producing, Zabnotto estimates, about 50,000 units per week, a number that will increase when less amount of the antigen is required to know the result.

Although this is a commendable finding, much remains to be done. In particular, the application of that antigen to, for example, a device similar to a pregnancy test that provides a result of “in 15 minutes”, he says. “It will serve to solve one of the biggest problems we have with Zika in Brazil: we do not know how many people are infected”, affirms Zanotto. This technique will also allow retrospective serologic tests of samples stored between 2014 and 2016, which could not be analyzed before due to the poor quality of available tests.

“It will serve to solve one of the biggest problems we have with Zika in Brazil: we do not know how many people are infected”

Brazilian Health Minister, Marcelo Castro, announced earlier this year the development of a rapid commercial test for Zika, but how long will it take until the test is available? The Instituto Butantán director replies that “the ministry responds politically, I respond technically and scientifically.” “If the test development would had been a technological issue now it would be resolved, but it was not, it was a scientific question: we did not know which was the technological path to take.”

But although science is acting fast, the urgency is high. In Rio de Janeiro, the Unidades de Pronto Atendimento or UPA, small emergency hospitals distributed around the city, are saturated every night by citizens who, following the recommendations of the #ZIKAZERO campaign located in buses and walls, go to the doctor at the slightest suspicion.

“There are logistical difficulties: patient care, collecting the material, separating solutions, or sample preparation in the laboratory”, says António Gomes Pinto, from the diagnostic reagents program of Bio-Manguinhos - a center depending on the Fundaçao Oswaldo Cruz - that is about to launch the first rapid serologic tests against Zika. “We have collaborated with an American company, Chembio, and we are already in the production stage, we hope to have it available in May to meet the demand of the Ministry of Health.”

António Gomes draws his next serology test against Zika / María Tapia Seoane.

“But it will not be a common rapid test; it counts with innovations we did not have before.” Gomes Pinto draws on a sheet a box the size of a credit card, more or less the size of the desired test. Above it he draws two parallel lines. “The device carries a miniature laboratory, two trials are performed in parallel, an immunoglobulin G (IgG) and IgM test, and we will have results in about 5 minutes, we will know not only if the patient is infected, but also the state of the infection.”

Later, the device could be used to detect dengue or chikungunya.

It seems incredible that two months ago, in January, a cohort of Senegalese scientists led by Alpha Amadou Sall, the Pasteur Institute in Dakar, were invited to Sao Paulo to teach Zanotto and his companions how to fight Zika. In just a few months, Brazilian researchers have gone from absolute ignorance of the disease to develop a specific serological diagnosis.

However, achieving this rapid diagnosis against Zika is not the final destination, but a station through which this train has to pass to reach the next stop: the development of a vaccine. A common enemy for specialists in disciplines as different as placentas, mosquitoes, and pediatrics to back together and give birth to another choral work.