Relapse cases among patients under malaria treatment is the winner of the Undergraduate Category of the 2016 Young Researcher Award

Researchers assessed 46 people submitted to malaria treatment who, afterwards, recurred of the diseases symptoms

Many factors have brought to light the relevance of malaria by Plasmodium vivax, as the propagation of parasites resistant to the available drugs. Aside from this, the concept of malaria by P. vivax as a benign disease changed due to the de ion of severe cases and even death. Finally, a latent form of the parasite living in the liver acts as a reservoir for the disease. These have been some problems controlling the disease. This was demonstrated by Ana Carolina Rios Silvinos study, awarded by the 2016 Young Researcher Award, under the Undergraduate category, during the 52nd Congress of the Brazilian Society of Tropical Medicine (MedTrop).

According to her work, a cause for relapse cases by patients under malaria treatment could be genetic mutation in the enzyme responsible for metabolizing primaquine, a drug used to combat latent forms of the parasite in the liver. The study was recently published on Plos One scientific journal.

Also joining the team are graduate student Gabriel Luíz Costa and researchers Cor Jesus Fontes, Luzia Helena Carvalho, Cristiana Ferreira Alves de Brito and Taís Nóbrega de Sousa, head of the research.

Find below, the interview to the Brazilian Society of Tropical Medicines press office:

BSTM: How did the idea of the project appear?

Ana Carolina Rios Silvino: One of the great challenges controlling malaria by P. vivax is eliminating the hypnozoites, forms of the parasite that remain latent in the liver and are eventually reactivated,  causing late relapses of the disease. In 2012, researchers proved that primaquine requires biotransformation by enzymes from the cytochrome P450 (CYP) complex, and that CYP2D6 is the key enzyme for the drugs activity in the liver. However, this enzyme is highly polymorphic and this genetic variability determines different metabolic phenotypes (increase, decrease of absence of enzymatic function) which considerably influences its pharmacokinetics differences.

When the projects idea occurred, there was a single work suggesting primaquine treatment failure due to CYP2D6 enzyme genetic variability could be related to relapse events by P. vivax. Although a possible connection between low-metabolic phenotypes and relapses was suggested, this work was conducted with a very small number of individuals (only four low enzyme metabolizing individuals), what raised the need of new studies in this field.

Within this context, we developed a project aiming to investigate whether relapse events could be explained, at least partially, by genetic differences in metabolizing primaquine by the CYP2D6 enzyme. Specifically, we assessed the frequency of polymorphisms, associated to a decrease of its function in two groups with different relapse incidences.

Besides this, we proposed to investigate, for the first time, if the failure treating malaria by P. vivax could be related to shifts metabolizing chloroquine. Currently, the first line therapy scheme for this species consists in an association of primaquine and chloroquine, which act in the liver and erythrocyte phases, respectively. Chloroquine is biotransformed mainly by the cytochrome enzyme P450 2C8 (CYP2C8). In this study, we raised the hypothesis that chloroquine therapy failure, associated to a reduced CYP2C8 metabolism, could contribute to malaria by P. vivax relapses.

BSTM: What did the work assess and how was the research conducted?

Ana Carolina Rios Silvino: In this pioneer study, we investigated if variants of the CYP2D6 enzyme were associated to an increased risk of relapses by P. vivax. For such, we studied five CYP2D6 enzyme polymorphisms responsible for a reduced or absent enzyme metabolic activity and two CYP2C8 polymorphisms determinant of a reduced chloroquine metabolic phenotype. The studys population consisted of 46 relapsing individuals.

The study was conducted with patient samples from Júlio Müller Hospital in Cuiabá/MT, considered a malaria reference center from 2004 to 2013. All patients selected for the research were diagnosed there, after travelling to endemic areas, where they were infected by P. vivax. After diagnosis and treatment, these people never returned to endemic areas Therefore, the new episodes of the disease were relapses and not new infections.

The selected individuals were split into two groups according to the number of relapses registered in six months: a group of individuals with a single relapse and a group of individuals with multiple relapses (two or three relapse episodes). After the selection, samples were genotyped by real-time PCR for polymorphisms in CYP2D6 and CYP 2C8.

While estimating the individuals genotypes frequencies for the studied polymorphisms, we observed a significantly larger frequency of people with the mutated allele in the group with multiple relapses. These findings proved our hypothesis that genetic variability in CYP2D6 can influence primaquine biotransformation, resulting in relapse episodes. In addition, we found a higher frequency of relapse parasites equivalent or similar to the initial parasites in patients with CYP2D6 polymorphisms.

In this study, we also assessed how these polymorphisms interfered in the CYP2D6 interaction with its substrate through an in silico analysis. This way, we proved the presence of enzyme polymorphism interferes with the CYP2D6 stability, and thus, the enzyme-primaquine interaction.

Finally, we demonstrated that CYP2C8 polymorphism associated to reduced chloroquine metabolism also contributes to P. vivax infection relapses. The occurrence of these polymorphisms was not different between both groups, since this drug does not act over the livers hypnozoites. However, individuals with polymorphisms in this enzyme relapsed earlier, suggesting difficulty eliminating the erythrocyte forms of the parasite by chloroquine.

BSTM: What is intended with this research?

Ana Carolina Rios Silvino:  The research aimed to assess whether CYP2D6 genetic variability, associated to slow and drug-free metabolism could influence primaquine biotransformation, leading to relapse episodes by P. vivax. Unlike primaquine, biotransformed chloroquine by CYP2C8 acts eliminating erythrocyte forms. This way, we also investigated if polymorphisms affecting CYP2C8 could contribute to malaria by P. vivax recurrences.

BSTM: How can your work contribute to the medical community and/or the society?

Ana Carolina Rios Silvino: Among the many factors affecting drug metabolism, interpersonal genetic variability can lead to changes in metabolism extension and drug elimination rate from the body. This feature is one of the reasons patients could differ their answers to anti-malaria treatment.

In our study, the great clinical relevance of CYP2D6 polymorphisms is related to a greater probability of adverse reactions, such as toxicity by drug buildup in the body and absence or low availability of the active compound that will effectively eliminate the parasite in its blood or liver form. This way, an individually adequate therapy, aiming to reduce adverse effects and increase the drug response effectiveness becomes extremely relevant.

Our results stress the importance of pharmacogenetics to monitor the effectiveness of anti-malaria therapy among patients infected by P. vivax, once we proved that part of the relapse cases are due to therapy errors in primaquine treatment. Within this context, the CYP2D6 enzyme pharmacogenetics contribution in primaquine biotransformation could reveal new horizons for the future of anti-malaria therapy, helping the medical community better understand those factors able to lead to therapy failure and evaluating patients who had relapses after primaquine treatment.

Generally speaking, establishing an adequate treatment, with effective posologies, where the drug is correctly metabolized and remains enough time in the body to release the desired effect: complete parasite elimination is a promising alternative and could help eliminating and controlling malaria by P. vivax.

BSTM: How many people were involved, among researchers and subjects?

Ana Carolina Rios Silvino: The research involved researchers and undergraduate and graduate students from the Malaria Molecular Biology and Immunology team at the René Rachou Research Center, Oswaldo Cruz Foundation (Fiocruz), in Belo Horizonte, Minas Gerais; besides researchers from the Biosystems Computer Science group at the René Rachou Research Center, Oswaldo Cruz Foundation (Fiocruz), in Belo Horizonte, Minas Gerais; from the Biochemistry Department at Cambridge University, Cambridge, England and Júlio Müller Hospital, Federal University of Mato Grosso, Cuiabá, Mato Grosso, Brazil. The study included analysis of 46 patients infected by P. vivax in endemic regions and who had relapses from 2004 to 2013.

BSTM: When was the research conducted?

Ana Carolina Rios Silvino: The research began in August 2014, and part of the data was published on July 28, 2016.

BSTM: Where?

Ana Carolina Rios Silvino: The experimental part of the research was conducted by the Malaria Molecular Biology and Immunology team at the René Rachou Research Center (Fiocruz/Minas).

Patient diagnosis and treatment were held at the Júlio Müller Teaching Hospital in Cuiabá (MT) from 2004 to 2013. The hospital is considered a reference center for malaria treatment and is located in a region currently considered in pre-elimination phase for malaria, where infections by P. vivax are considered highly improbable.

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