In response to the growth in cases of yellow fever, the Ministry of Health has distributed, since 2017, more than 68 million doses of the vaccine to Brazilian states – 52 million doses more than the total distributed in 2016.
In March 2018, the Ministry announced that the entire national territory will be a vaccine recommendation area by April 2019, with a forecast of immunizing more than 77 million people. A central measure for disease prevention and control, the vaccine is considered safe and is 95% to 99% effective. However, like any vaccine or medicine, it can cause adverse events.
In these rare cases, symptoms identical to those of natural infection with the virus occur. To distinguish the origin of the case, it is necessary to identify, in the laboratory, whether the patient has the wild virus – the one circulating in a given location, transmitted by mosquito bites – or the attenuated virus – which is used in the production of the vaccine.
An innovation in molecular diagnosis conceived by the Instituto Oswaldo Cruz (IOC/Fiocruz) and developed in partnership with the University of Bonn, Germany, allows to differentiate with precision and more agility if the origin of the case was a common transmission or an adverse event after vaccination.
The novelty was developed by the Flavivirus Laboratory of IOC, which acts as a regional reference for yellow fever with the Ministry of Health.
Virologist Ana Bispo conceived the project based on a concrete demand.
“We created a solution that allows us to provide faster responses in terms of diagnosis. In the face of an outbreak of yellow fever, laboratory confirmation is an important tool in defining strategies for surveillance of virus circulation and disease control,” said the head of the Flavivirus Laboratory.
The differential method, which uses the RT-PCR technique in real time, greatly surpasses the speed of the traditional virus genetic sequencing technique, currently available for differentiating between wild virus and vaccine virus. While sequencing can range from three to 15 days, the new real-time RT-PCR protocol takes one to two hours.
In addition, it can be conducted by a professional who masters the basic techniques of molecular diagnosis, while the viral sequencing method requires professionals with specific training to analyze the results of sequencing the genetic material of the virus.
The development and validation of the protocol are described in an article published in the scientific journal 'Emerging Infectious Diseases', published by the US Center for Disease Control and Prevention (CDC).
"We created a solution that allows us to provide faster answers in terms of diagnosing yellow fever", highlights virologist Ana Bispo, one of the leaders of the research. Photo: Gutemberg Brito
The real-time RT-PCR methodology is based on identifying the genetic material of the virus in a sample. To arrive at an accurate test, the researchers looked for regions of the genome in which the wild virus and the vaccine strain are different, which allows the differentiation between both.
Two protocols were created: the so-called 'single target' protocol (when there is a need to carry out two separate reactions to detect the presence of the wild virus and the vaccine strain) and the 'dual target' protocol (when the detection is performed in the same reaction).
The protocol proved to be capable of detecting the wild virus and the vaccine virus with high diagnostic sensitivity and specificity.
“One point deserves emphasis: to ensure that there was no risk of cross-reaction, experiments were carried out with more than 40 different viruses during the development of the method. We were pleasantly surprised to be able to develop an unprecedented method of high sensitivity, specificity and rapidity to differentiate wild yellow fever virus and vaccine virus”, celebrates the researcher.
In addition, the procedure allows quantifying the viral load present in the sample.
Currently, the procedure is being applied at the Flavivirus Laboratory of IOC to characterize the type of infection in samples of suspected cases of adverse events after vaccination.
The protocol has the potential to contribute especially in the routine of clarifying suspected cases of adverse events to vaccination with the National Immunization Program (PNI). At the request of the General Coordination of Public Health Laboratories of the Ministry of Health (CGLAB), the production, on a prototype basis, of a kit of supplies for use in the protocol is underway.
This step is being carried out in collaboration with the IOC with the Institute of Molecular Biology of Paraná (IBMP). The perspective is that, in the future, the inputs can be used in the network of Central Public Health Laboratories (Lacens), which operates in the laboratory diagnosis of yellow fever. For André Luiz de Abreu, from the General Coordination of Public Health Laboratories of the Ministry of Health (CGLAB/SVS/MS), the novelty could benefit the work routine across the country.
“This is an innovation devised by a Laboratory in the network to improve the set of differentiating activities in diagnosing yellow fever. There is potential to streamline the process across Brazil,” he said.
The procedure is being applied at the Flavivirus Laboratory of IOC to characterize the type of infection in samples of suspected cases of adverse events after vaccination. Photo: Gutemberg Brito
“Like the unprecedented idealization of the development of a rapid and sensitive test capable of simultaneously diagnosing Zika, dengue and chikungunya and now available in the Lacens network, the new proposal developed at the Flavivirus Laboratory represents yet another important achievement for laboratory diagnosis. Once again, we were able to obtain a concrete solution to a routine challenge for laboratories that deal with the diagnosis of these diseases”, the virologist details.
The new protocol has one more benefit: it offers conclusive results even when the sample has a low concentration of the virus. In these cases, it may not be possible to perform the traditional analysis directly from the clinical material, requiring an intermediate step to isolate the virus, which is then replicated in the laboratory.
However, the situation becomes a problem in the hypothesis of a patient who has both the wild and the vaccine infection - a patient, therefore, who is bitten by a mosquito with the virus shortly before or shortly after the moment when he was vaccinated, when immunity provoked by vaccination has not yet been established.
In this circumstance, the chances increase that, due to the intermediate stage of viral isolation, only the virus present in greater quantity in the sample will be detected, despite the two viruses being present. The new differential real-time RT-PCR protocol is capable of detecting the presence of both viruses at the same time – even if one of them is at a lower concentration in the patient's sample.
The Flavivirus Laboratory of the Oswaldo Cruz Institute (IOC/Fiocruz) works regularly since the 1990s in the laboratory diagnosis of samples of patients with suspected disease from the states of Rio de Janeiro, Espírito Santo, Minas Gerais and Bahia.
It has also carried out analyzes of primate samples since 2014.
In addition, with the increase in the number of suspected cases since 2017, the laboratory was also designated by the Ministry of Health to process samples from Ceará and Rio Grande do Norte. According to the Ministry of Health, between June 2016 and June 2017, 777 cases and 261 deaths from yellow fever were confirmed in the country. And from July 2017 to April 03, 2018, 1.127 cases and 328 deaths have already been recorded.
In response to the growth in cases of yellow fever, the Ministry of Health has distributed, since 2017, more than 68 million doses of the vaccine to Brazilian states – 52 million doses more than the total distributed in 2016.
In March 2018, the Ministry announced that the entire national territory will be a vaccine recommendation area by April 2019, with a forecast of immunizing more than 77 million people. A central measure for disease prevention and control, the vaccine is considered safe and is 95% to 99% effective. However, like any vaccine or medicine, it can cause adverse events.
In these rare cases, symptoms identical to those of natural infection with the virus occur. To distinguish the origin of the case, it is necessary to identify, in the laboratory, whether the patient has the wild virus – the one circulating in a given location, transmitted by mosquito bites – or the attenuated virus – which is used in the production of the vaccine.
An innovation in molecular diagnosis conceived by the Instituto Oswaldo Cruz (IOC/Fiocruz) and developed in partnership with the University of Bonn, Germany, allows to differentiate with precision and more agility if the origin of the case was a common transmission or an adverse event after vaccination.
The novelty was developed by the Flavivirus Laboratory of IOC, which acts as a regional reference for yellow fever with the Ministry of Health.
Virologist Ana Bispo conceived the project based on a concrete demand.
“We created a solution that allows us to provide faster responses in terms of diagnosis. In the face of an outbreak of yellow fever, laboratory confirmation is an important tool in defining strategies for surveillance of virus circulation and disease control,” said the head of the Flavivirus Laboratory.
The differential method, which uses the RT-PCR technique in real time, greatly surpasses the speed of the traditional virus genetic sequencing technique, currently available for differentiating between wild virus and vaccine virus. While sequencing can range from three to 15 days, the new real-time RT-PCR protocol takes one to two hours.
In addition, it can be conducted by a professional who masters the basic techniques of molecular diagnosis, while the viral sequencing method requires professionals with specific training to analyze the results of sequencing the genetic material of the virus.
The development and validation of the protocol are described in an article published in the scientific journal 'Emerging Infectious Diseases', published by the US Center for Disease Control and Prevention (CDC).
"We created a solution that allows us to provide faster answers in terms of diagnosing yellow fever", highlights virologist Ana Bispo, one of the leaders of the research. Photo: Gutemberg Brito
The real-time RT-PCR methodology is based on identifying the genetic material of the virus in a sample. To arrive at an accurate test, the researchers looked for regions of the genome in which the wild virus and the vaccine strain are different, which allows the differentiation between both.
Two protocols were created: the so-called 'single target' protocol (when there is a need to carry out two separate reactions to detect the presence of the wild virus and the vaccine strain) and the 'dual target' protocol (when the detection is performed in the same reaction).
The protocol proved to be capable of detecting the wild virus and the vaccine virus with high diagnostic sensitivity and specificity.
“One point deserves emphasis: to ensure that there was no risk of cross-reaction, experiments were carried out with more than 40 different viruses during the development of the method. We were pleasantly surprised to be able to develop an unprecedented method of high sensitivity, specificity and rapidity to differentiate wild yellow fever virus and vaccine virus”, celebrates the researcher.
In addition, the procedure allows quantifying the viral load present in the sample.
Currently, the procedure is being applied at the Flavivirus Laboratory of IOC to characterize the type of infection in samples of suspected cases of adverse events after vaccination.
The protocol has the potential to contribute especially in the routine of clarifying suspected cases of adverse events to vaccination with the National Immunization Program (PNI). At the request of the General Coordination of Public Health Laboratories of the Ministry of Health (CGLAB), the production, on a prototype basis, of a kit of supplies for use in the protocol is underway.
This step is being carried out in collaboration with the IOC with the Institute of Molecular Biology of Paraná (IBMP). The perspective is that, in the future, the inputs can be used in the network of Central Public Health Laboratories (Lacens), which operates in the laboratory diagnosis of yellow fever. For André Luiz de Abreu, from the General Coordination of Public Health Laboratories of the Ministry of Health (CGLAB/SVS/MS), the novelty could benefit the work routine across the country.
“This is an innovation devised by a Laboratory in the network to improve the set of differentiating activities in diagnosing yellow fever. There is potential to streamline the process across Brazil,” he said.
The procedure is being applied at the Flavivirus Laboratory of IOC to characterize the type of infection in samples of suspected cases of adverse events after vaccination. Photo: Gutemberg Brito
“Like the unprecedented idealization of the development of a rapid and sensitive test capable of simultaneously diagnosing Zika, dengue and chikungunya and now available in the Lacens network, the new proposal developed at the Flavivirus Laboratory represents yet another important achievement for laboratory diagnosis. Once again, we were able to obtain a concrete solution to a routine challenge for laboratories that deal with the diagnosis of these diseases”, the virologist details.
The new protocol has one more benefit: it offers conclusive results even when the sample has a low concentration of the virus. In these cases, it may not be possible to perform the traditional analysis directly from the clinical material, requiring an intermediate step to isolate the virus, which is then replicated in the laboratory.
However, the situation becomes a problem in the hypothesis of a patient who has both the wild and the vaccine infection - a patient, therefore, who is bitten by a mosquito with the virus shortly before or shortly after the moment when he was vaccinated, when immunity provoked by vaccination has not yet been established.
In this circumstance, the chances increase that, due to the intermediate stage of viral isolation, only the virus present in greater quantity in the sample will be detected, despite the two viruses being present. The new differential real-time RT-PCR protocol is capable of detecting the presence of both viruses at the same time – even if one of them is at a lower concentration in the patient's sample.
The Flavivirus Laboratory of the Oswaldo Cruz Institute (IOC/Fiocruz) works regularly since the 1990s in the laboratory diagnosis of samples of patients with suspected disease from the states of Rio de Janeiro, Espírito Santo, Minas Gerais and Bahia.
It has also carried out analyzes of primate samples since 2014.
In addition, with the increase in the number of suspected cases since 2017, the laboratory was also designated by the Ministry of Health to process samples from Ceará and Rio Grande do Norte. According to the Ministry of Health, between June 2016 and June 2017, 777 cases and 261 deaths from yellow fever were confirmed in the country. And from July 2017 to April 03, 2018, 1.127 cases and 328 deaths have already been recorded.
The non-profit reproduction of the text is allowed as long as the source is cited (Comunicação / Instituto Oswaldo Cruz)
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