Plasmodium vivax is the most geographically widespread species causing human malaria, with approximately 40% of the world's population at risk of infection 1–3. There was near 157,000 new cases in Brazil in 2019, most due P. vivax (89.1%), of which around 21% being classified as recurrences within 60 days 4. Relapses, that accounted for around 33.000 episodes that year, and earlier gametocyte production make this species especially challenging for treatment and control, but the mechanisms leading to activating hypnozoites remain unknown5.
Radical cure of vivax malaria requires antimalarial drugs that target both blood and liver stages. Primaquine (PQ) is the most available drug to eliminate hypnozoites6,7. PQ’s clinical effectiveness is limited by the toxicity and potential hemolytic adverse events in patients with glucose-6 phosphate deficiency (G6PD) that is why, the drug is contraindicated during pregnancy and for infants less than six months.
There is no definitive method to differentiate recurrences of P. vivax as recrudescence, especially with increasing evidence of resistance to chloroquine (CQ)8, relapses, and reinfection in areas with active transmission. The possibility of following patients in non-endemic areas provides an advantage where at least reinfection could be excluded. Recent studies in Brazil showed that recurrences rates in the Amazon setting range from 29.4% to 39.6% in Amazon and non-Amazon areas9–11, despite PQ’s routine prescription. It was recently described that cytochrome P450 2D6 (CYP2D6) pathway mediates the activation of primaquine into active phenolic metabolite(s) in hepatocytes12,13 and some genetic polymorphisms implied in reduced PQ metabolism have been associated with higher risk of relapse14. Individuals with specific CYP2D6 polymorphic alleles fail to metabolize PQ and may experience treatment failure, leading to false assumptions of PQ efficacy and tolerance.
CYP2D6 gene is highly polymorphic with over 150 alleles categorized in no, decreased, normal, and increased function alleles based on enzyme activity15. The CYP2D6 allele combinations give rise to different predicted metabolizer phenotypes: poor (gPM), intermediate (gIM), normal (gNM), and ultrarapid (gUM) metabolizers15. Therefore, vivax malaria patients with the defective CYP2D6 function would be at increased risk for therapeutic failure (relapses) regardless of proper treatment regimens with PQ14,16–18. Thereat, to identify patients at a higher risk for recurrences CYP2D6 metabolizer status provides valuable information in improving the interpretation of treatment failure in P. vivax and strengthening the efforts to control this parasite. Herein, we describe three cases of multiple vivax malaria relapses in individuals with impaired CYP2D6 metabolic activity followed-up at a non-endemic area in Brazil. The individual responses to different drug schemes varied related to CYP2D6 metabolic status and showed to be complex.
Site and standard procedures:
The Instituto Nacional de Infectologia Evandro Chagas (INI) is a reference center for diagnosing and treating infectious diseases at Fundação Oswaldo Cruz (Fiocruz), in Rio de Janeiro, Brazil. Patients with suspicion of malaria are evaluated by infectious disease physicians and follow the national malaria treatment guidelines. The guidelines state that vivax malaria should be treated with CQ (25mg/kg during three days) and PQ (3.5mg/kg during seven or 14 days). Blood slides were collected by experienced microscopists and malaria species confirmed by polymerase chain reaction (PCR) 19,20. Patients were followed until parasitological clearance and routinely at days 3, 7, 14, 21, 28, 40, and 60 post-treatment and at any time in case of recurring fever. All patients were tested for G6PDd. PQ was adjusted for body weight (bw) when necessary. None of the patients returned to the endemic area.
Genotyping of one tri-nucleotide deletion (2615-2617delAAG [rs5030656]), eight single-nucleotide polymorphisms (SNPs) (-1584C>G [rs1080985], 100C>T [rs1065852], 1023C>T [rs28371706], 1846G>A [rs3892097], 2850C>T [rs16947], 2988G>A [rs28371725], 3183G>A [rs59421388], 4180G>C [rs1135840]) and CYP2D6 copy number analysis were performed by Real-Time PCR, according to protocols previously described16,22. CYP2D6 haplotypes were inferred from genotypes using the software PHASE v.2.123,24 and phenotypes were predicted based on activity score (AS) model 15. Patients were categorized into five predicted phenotype classes: poor metabolizer (gPM; AS score = 0), intermediate metabolizer (gIM; AS score = 0.5), normal-slow metabolizer (gNM-S; AS score = 1), normal-fast metabolizer (gNM-F; AS score 1.5-2.0), and ultrarapid metabolizer (gUM ; AS score > 2).
Ethical approval was obtained from the INI-Fiocruz Ethical Board (number 0020.0.009.000-07), and all participants provided informed written consent.