Samples were systematically collected from women who showed symptoms of the disease during pregnancy; of 17 cases investigated, 14 tested positive
Research on placentas from women who had children at Caism (Center for Comprehensive Attention to Women's Health) at Unicamp identified 14 positive cases for Zika virus, among 17 analyzed, showing that the placenta is an important reservoir for the virus in infection. Pregnant women who presented symptoms of the disease – fever and spots on the skin (febrile exanthematous lesions) – were monitored during pregnancy or with fetuses with microcephaly, in the years 2016 and 2017. Tissue analysis was carried out after systematic collection following the Biobank protocol. institution. However, the test carried out at the reference laboratory, using the protocol recommended by the Ministry of Health, was negative in all of these cases. Of the total of 77 women identified with symptoms, 49 gave birth at Caism. There were two cases of microcephaly, both resulting in neonatal death.
“It was a surprising result, which brings with it the need to look for mothers whose placenta tests were positive, and these children, to carry out a complete evaluation and observe whether complications related to the Zika virus that were not identified at the time of birth did not arise, despite the strict evaluation and monitoring that were then recommended”, says professor Eliana Amaral, from the Faculty of Medical Sciences (FCM), who supervised the study financed by Fapesp and CNPq. “Microcephaly is the pinnacle in terms of severity, but other complications – neurological, ocular or auditory – can occur. After the births, we scheduled return visits for follow-up by specialists, but with the absence of apparent problems, few mothers returned with their children.”
Another reason for this low return would be the mothers' lack of concern regarding the negative results of serology (blood) and urine tests, which were collected when they showed symptoms of the disease during pregnancy – which can happen due to the short duration of positive tests for this infection – but This behavior should change when they learn of the positive result of the placenta exam. “The placenta is always an organ to study in cases of infection and, in the case of Zika, it proved to be a marker that the mother had the disease, that the virus, at least, reached there, and could have infected the child or been a barrier”, attests the obstetrics professor at the Faculty of Medical Sciences (FCM).
The laboratory tests were carried out by Emanuella Meneses Venceslau in her doctoral thesis, which was co-supervised by professor Maria Laura Costa do Nascimento, from FCM, and in collaboration with José Luiz Proença Módena, coordinator of the Emerging Virus Laboratory at the Institute of Biology ( LEVE-IB). “The Biobank protocol recommends systematic collection, in various regions of the placenta (chorionic plate, chorionic villus, basal plate), in order to guarantee adequate representation, in addition to the amniotic membrane and the umbilical cord. The 14 placentas were positive by the RT-qPCR kit for ZIKV used in our research laboratory, compared to none of the samples coming from a simple collection per placenta and analyzed in the reference laboratory, with another kit”, explains the author of the thesis. . “The use of the same kit recommended by the standard laboratory was able to identify 10 of the 14 positive cases using the research kit. In other words, probably the biggest difference is related to the standard of sample collection, storage and transportation.”
Emanuella Venceslau recalls that the Zika outbreak that occurred between April 2015 and November 2016 is considered the largest of this type of virus in history. “After this peak occurred in Brazil, it spread to other countries in South America, Central America and the Caribbean. Despite the drop in the number of cases, infection with the Zika virus continues to be a concern for pregnant women, as stated by infectologist Artur Timermann, president of the Brazilian Society of Arborivores [diseases transmitted by insects].”
Government agility
In Eliana Amaral's opinion, at the beginning of the epidemic in Brazil, the government sought to provide the quickest responses possible at the time, establishing a protocol for exams and creating the infrastructure for transporting and analyzing samples – the reference laboratory in the state of São Paulo is the Adolfo Lutz Institute. “Unicamp, in turn, was immediately concerned about the vertical transmission of the disease (from pregnant women to children) and created a collaborative research group initially led by IB, but involving the health units, Caism and Hospital das Clínicas. It was also the time when Caism created its placenta Biobank, storing samples with authorization, to be able to carry out further studies on various diseases, including infections and others that can affect pregnant women and their children.”
The research advisor credits professor Maria Laura Costa with the proposal to adopt a protocol that recommends obtaining samples from different places in the placenta, based on experience she had during her postdoctoral studies at Washington University, in Saint Louis (USA). “This is the first research that demonstrates the need to change the method of collection and adopt the test kit that showed greater positivity for the Zika virus.”
Maria Laura Costa highlights the great importance of the placenta for the diagnosis, which is difficult to define in the acute phase of the disease. “Many patients are asymptomatic, others have mild symptoms and there are those who delay seeking health services. And the window of opportunity for a test to be positive, in acute infection, is small: up to five days for blood and up to eight days for urine. The placenta is an organ where positivity remains for months and months after infection.”
Researcher José Módena adds that 70% of those infected with the Zika virus are asymptomatic and that, even focusing on the other 30%, the symptoms are broad and mild, causing the patient to delay seeking care. “Sometimes, when he searches, the window of opportunity for diagnosis has already expired. An alternative for several infectious diseases is to look for the antibody, to find out if the person has already come into contact with that pathogen. There are commercial kits for serological analysis, but we are in a country that has many viruses similar to Zika, such as dengue and yellow fever. There is a very high cross-reactivity, making diagnosis after the acute phase difficult.”
For Módena, the Unicamp group created the opportunity to obtain a differentiated placenta sample, collected in a stratified, systematized manner and monitored from collection to storage. “In the study, there was full quality and temperature control, making it possible to evaluate the detection of viruses in the placenta as an indication to make an accurate diagnosis in mothers who presented symptoms at some point during pregnancy or who had some suggestive fetal complication of congenital zika, from mother to child. Another characteristic of Zika virus infection is that, if the pregnant woman comes into contact with the agent, she does not always produce antibodies, which makes the diagnosis even more challenging.”
Collection and storage
Maria Laura Costa explains that preserving placentas is not a standard procedure and that they are normally discarded. “It is very difficult to work with samples collected at the time of birth, because most of the time it is not scheduled and carried out by a team on duty. If the placenta is an organ that weighs an average of 400 grams, it is important to reflect on how representative just one sample can be. Therefore, team training is essential to guarantee the representativeness and quality of the samples. Sample collection must be as early as possible, with a time limit of up to 12 hours. In our study, collection took place in a very short period of time, up to two hours after birth. We took samples from four different regions of the placenta, to be representative, and immediately placed the tissue in liquid nitrogen (at minus 80ºC), which guarantees quality for the exam.”
José Módena informs that, according to the official standardized protocol, there is no obligation to send the sample at low temperature to the reference laboratory, which is centralized in São Paulo. “It is recommended, but most centers are unable to comply with these transport conditions. The protocol only describes that the sample must measure 1 centimeter by 1 centimeter, without specifying a region of the placenta, nor the time or quality of transport and storage.”
Professor Eliana Amaral highlights the need, now, to talk to health authorities to suggest that the standardization be changed. “The official protocol in force across the country certainly did not focus on these technical details, especially because there were no research results to show that this should be the case. There is also the issue of the kits used, since ours was different from the one adopted at the time by the reference laboratory. The channel with the authorities is always there because Unicamp is a reference in the care of pregnant women. We have already carried out several studies that led to changes in behavior in the state of São Paulo, for example, when there was mass vaccination against yellow fever or rubella and pregnant women were inadvertently vaccinated. We showed that nothing happened, that the vaccine was safe. We are already recognized for this expertise, which is our role: to carry out research that can improve people’s health care.”
Unicamp researchers reinforce that the intention is to collaborate with the bodies that define health policies, praising the speed with which researchers and the government, together, sought to organize initial care for victims of the Zika virus epidemic. But they emphasize that the improvement of protocols based on new research is a routine collaboration between the University and health bodies, especially in the areas of infections during pregnancy, since the end of the 80s. “The issue here is to highlight the difference in results in detecting the virus. It is not a question of identifying a quality problem, because perhaps the sample collected according to the recommendations then envisaged would not even allow detection”, observes José Módena.
Maria Laura Costa warns that, in addition to the Zika virus, other emerging viruses are constant threats. “They are there and at any moment we can have a new epidemic, a new virus, and we must be prepared for a quick response. On Zika, long-term follow-up studies are beginning; No one knows for sure what the outcome would be like for babies born without serious malformations, but to mothers whose placenta tests were positive. They are very young and we still need to define whether they have sequelae or not. The new phase of this study, reevaluating these children, will help to find some of the answers to the remaining questions.”
Cases of probable Zika infection (Brazil, 2019)
