Influence of CYP2C8, CYP3A4, and CYP3A5 Host Genotypes on Early Recurrence of Plasmodium vivax.

Cytochrome P450 (CYP) enzymes are involved in the biotransformation of chloroquine (CQ), but the role of the different profiles of metabolism of this drug in relation to Plasmodium vivax recurrences has not been properly investigated. To investigate the influence of the CYP genotypes associated with CQ metabolism on the rates of P. vivax early recurrences, a case-control study was carried out. The cases included patients presenting with an early recurrence (CQ-recurrent individuals), defined as a recurrence during the first 28 days after initial infection and plasma concentrations of CQ plus desethylchloroquine (DCQ; the major CQ metabolite) higher than 100 ng/ml. A control group with no parasite recurrence over the follow-up (the CQ-responsive group) was also included. CQ and DCQ plasma levels were measured on day 28. CQ-metabolizing CYP (CYP2C8, CYP3A4, and CYP3A5) genotypes were determined by real-time PCR. An ex vivo study was conducted to verify the efficacy of CQ and DCQ against P. vivax isolates. The frequency of alleles associated with normal and slow metabolism was similar between the cases and the controls for the CYP2C8 (odds ratio [OR] = 1.45, 95% confidence interval [CI] = 0.51 to 4.14, P = 0.570), CYP3A4 (OR = 2.38, 95% CI = 0.92 to 6.19, P = 0.105), and CYP3A5 (OR = 4.17, 95% CI = 0.79 to 22.04, P = 1.038) genes. DCQ levels were higher than CQ levels, regardless of the genotype. Regarding the DCQ/CQ ratio, there was no difference between groups or between those patients who had a normal genotype and those patients who had a mutant genotype. DCQ and CQ showed similar efficacy ex vivo CYP genotypes had no influence on early recurrence rates. The similar efficacy of CQ and DCQ ex vivo could explain the absence of therapeutic failure, despite the presence of alleles associated with slow metabolism.

TOLLIP gene variant is associated with Plasmodium vivax malaria in the Brazilian Amazon.

BACKGROUND: Toll-interacting protein is a negative regulator in the TLR signaling cascade, particularly by impeding the TLR2 and, TLR4 pathway. Recently, TOLLIP was shown to regulate human TLR signaling pathways. Two common TOLLIP polymorphisms (rs5743899 and rs3750920) were reported to be influencing IL-6, TNF and IL-10 expression. In this study, TOLLIP variants were investigated to their relation to Plasmodium vivax malaria in the Brazilian Amazon. METHODS: This cohort study was performed in the municipalities of Careiro and, Manaus, in Western Brazilian Amazon. A total of 319 patients with P. vivax malaria and, 263 healthy controls with no previous history of malaria were included in the study. Genomic DNA was extracted from blood collected on filter paper, using the QIAamp® DNA Mini Kit, according to the manufacturer's suggested protocol. The rs5743899 and rs3750920 polymorphisms of the TOLLIP gene were typed by PCR-RFLP. RESULTS: Homozygous individuals for the rs3750920 T allele gene had twice the risk of developing malaria when compared to individuals homozygous for the C allele (OR 2.0 [95% CI 1.23-3.07]; p = 0.004). In the dominant model, carriers the C allele indicates protection to malaria, carriers of the C allele were compared to individuals with the T allele, and the difference is highly significant (OR 0.52 [95% CI 0.37-0.76]; p = 0.0006). The linkage disequilibrium between the two polymorphisms was weak (r2 = 0.037; D' = 0.27). CONCLUSIONS: These findings suggest that genes involved in the TLRs-pathway may be involved in malaria susceptibility. The association of the TOLLIP rs3750920 T allele with susceptibility to malaria further provides evidence that genetic variations in immune response genes may predispose individuals to malaria.

Association of CYP2C19, CYP2D6 and CYP3A4 Genetic Variants on Primaquine Hemolysis in G6PD-Deficient Patients.

In the Amazon, the treatment for Plasmodium vivax is chloroquine plus primaquine. However, this regimen is limited due to the risk of acute hemolytic anemia in glucose-6-phosphate dehydrogenase deficiency. Primaquine is a prodrug that requires conversion by the CYP2D6 enzyme to be effective against malaria. A series of cases were performed at an infectious diseases reference hospital in the Western Brazilian Amazon. The STANDARD G6PD (SD Biosensor®) assay was used to infer G6PD status and real-time PCR to genotype G6PD, CYP2C19, CYP2D6 and CYP3A4. Eighteen patients were included, of which 55.6% had African A- variant (G202A/A376G), 11.1% African A+ variant (A376G), 5.6% Mediterranean variant (C563T) and 27.8% were wild type. CYP2C19, CYP2D6 and CYP3A4 genotyping showed no statistically significant differences in the frequency of star alleles between the groups G6PD deficient and G6PD normal. Elevated levels of liver and kidney markers in the G6PDd patients were observed in gNM, gRM and gUM of CYP2C19 and CYP2D6 (p < 0.05). Furthermore, in this study there was no influence of CYPs on hemolysis. These findings reinforce the importance of studies on the mapping of G6PD deficiency and genetic variations of CYP2C19, CYP2D6 and CYP3A4. This mapping will allow us to validate the prevalence of CYPs and determine their influence on hemolysis in patients with malaria, helping to decide on the treatment regimen.

Real-life implementation of a G6PD deficiency screening qualitative test into routine vivax malaria diagnostic units in the Brazilian Amazon (SAFEPRIM study).

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency greatly hinders Plasmodium vivax malaria radical cure and further elimination due to 8-aminoquinolines-associated hemolysis. Although the deleterious health effects of primaquine in G6PD deficient individuals have been known for over 50 years, G6PD testing is not routinely performed before primaquine treatment in most P. vivax endemic areas. METHOD/PRINCIPAL FINDINGS: The qualitative CareStart G6PD screening test was implemented in 12 malaria treatment units (MTUs) in the municipality of Rio Preto da Eva, Western Brazilian Amazon, a malaria endemic area, between February 2019 and early January 2020. Training materials were developed and validated; evaluations were conducted on the effectiveness of training health care professionals (HCPs) to perform the test, the interpretation and reliability of routine testing performed by HCPs, and perceptions of HCPs and patients. Most HCPs were unaware of G6PD deficiency and primaquine-related adverse effects. Most of 110 HCPs trained (86/110, 78%) were able to correctly perform the G6PD test after a single 4-hour training session. The test performed by HCPs during implementation showed 100.0% (4/4) sensitivity and 68.1% (62/91) specificity in identifying G6PD deficient patients as compared to a point-of-care quantitative test (Standard G6PD). CONCLUSIONS/SIGNIFICANCE: G6PD screening using the qualitative CareStart G6PD test performed by HCPs in MTUs of an endemic area showed high sensitivity and concerning low specificity. The amount of false G6PD deficiency detected led to substantial loss of opportunities for radical cure.

An Ultra-Sensitive Technique: Using Pv-mtCOX1 qPCR to Detect Early Recurrences of Plasmodium vivax in Patients in the Brazilian Amazon.

BACKGROUND: Early recurrence of Plasmodium vivax is a challenge for malaria control in the field, particularly because this species is associated with lower parasitemia, which hinders diagnosis and monitoring through blood smear testing. Early recurrences, defined as the persistence of parasites in the peripheral blood despite adequate drug dosages, may arise from resistance to chloroquine. The objective of the study was to estimate early recurrence of P. vivax in the Brazilian Amazon by using a highly-sensitive detection method, in this case, PCR. METHODS: An ultra-sensitive qPCR that targeted mitochondrial DNA was used to compare a standard qPCR that targeted 18S rDNA to detect early recurrence of P. vivax in very low densities in samples from patients treated with chloroquine. RESULTS: Out of a total of 312 cases, 29 samples (9.3%) were characterized as recurrences, from which 3.2% (10/312) were only detected through ultra-sensitive qPCR testing. CONCLUSIONS: Studies that report the detection of P. vivax early recurrences using light microscopy may severely underestimate their true incidence.