Tafenoquine (TQ) and primaquine (PQ) are 8-aminoquinolines (8-AQ) with anti-hypnozoite activity against vivax malaria. PQ is the only FDA-approved medicine for preventing relapsing Plasmodium vivax infection and TQ is currently in phase 3 clinical trials for the same indication. Recent studies have provided evidence that cytochrome P450 (CYP) metabolism via CYP2D6 plays a role in PQ efficacy against P. vivax and have suggested that this effect may extend to other 8-AQs, including TQ. Here, a retrospective pharmacogenetic (PGx) investigation was performed to assess the impact of CYP2D6 metabolism on TQ and PQ efficacy in the treatment of P. vivax in the DETECTIVE study (TAF112582), a recently completed, randomized, phase 2b dose-ranging clinical trial. The impact of CYP2D6 on TQ pharmacokinetics (PK) was also investigated in TAF112582 TQ-treated subjects and in vitro CYP metabolism of TQ was explored. A limitation of the current study is that TAF112582 was not designed to be well powered for PGx, thus our findings are based on TQ or PQ efficacy in CYP2D6 intermediate metabolizers (IM), as there were insufficient poor metabolizers (PM) to draw any conclusion on the impact of the PM phenotype on efficacy. The impact of genetically-predicted CYP2D6 reduced metabolism on relapse-free efficacy six months post-dosing of TQ or PQ, both administered in conjunction with chloroquine (CQ), was assessed using exact statistical methods in 198 P. vivax-infected study participants comparing IM to extensive metabolizers (EM). The influence of CYP2D6 metabolizer phenotypes on TQ PK was assessed comparing median TQ area under the curve (AUC). In vitro metabolism of TQ was investigated using recombinant, over-expressed human CYP enzymes and human hepatocytes. Metabolite identification experiments were performed using liquid chromatography-mass spectrometry. Reduction of CYP2D6 activity was not associated with an increase in relapse-rate in TQ-treated subjects (p = 0.57). In contrast, and in accordance with recent literature, CYP2D6 IMs were more common (p = 0.05) in PQ-treated subjects who relapsed (50 %) than in subjects who remained relapse-free (17 %). Further, CYP2D6 metabolizer phenotypes had no significant effect on TQ AUC, and only minimal metabolism of TQ could be detected in hepatic in vitro systems. Together, these data provide preliminary evidence that in CYP2D6 IMs, TQ efficacy in P. vivax-infected individuals is not diminished to the same extent as PQ. As there were no PMs in either the TQ or PQ treatment arms of TAF112582, no conclusions could be drawn on potential differences in PMs. These findings suggest that differential effects of CYP2D6 metabolism on TQ and PQ efficacy could be a differentiation factor between these 8-AQs, but results remain to be confirmed prospectively in the ongoing phase 3 studies.

Galectin-9 (Gal-9) is a β-galactoside-binding lectin that interacts with sugar moieties on glycoproteins and glycolipids of cells and pathogens. Gal-9 is known as an immune modulator that induces cell death via interaction with T cell immunoglobulin and mucin domain-3 (Tim3), a co-inhibitory receptor, and it inhibits production of several pro-inflammatory cytokines (TNF, IL-6 and IL-1α) and enhances production of IL-10. To understand the immune pathology of malaria, the Gal-9 in plasma was measured. Plasma samples and clinical parameters were obtained from 50 acute malaria cases (nine severe and 41 uncomplicated cases) from Thailand at three time points: day 0, day 7 and day 28. Gal-9 levels were determined by ELISA. A total of 38 species of cytokines and chemokines were measured using a BioPlex assay. Gal-9 levels were higher at day 0 compared to day 7 and day 28 (P < 0.0001). Gal-9 levels were also higher in severe malaria (SM) cases compared to uncomplicated (UM) cases at day 0 and day 7 (923 vs 617 pg/mL; P = 0.03, and 659 vs 348 pg/mL; P = 0.02 respectively). Median Gal-9 levels were higher in patients with blood urea nitrogen to creatinine ratio (BUN/creatinine) ≥20 (mg/dL) than in patients with BUN/creatinine <20 (mg/dL) at day 0 (817.3 vs 576.2 pg/mL, P = 0.007). Gal-9 was inversely significantly correlated with chloride levels in both SM and UM cases (r s = −0.73 and r s = −0.46, respectively). In both UM and SM cases, Gal-9 was significantly associated with pro- and anti-inflammatory cytokines and chemokines such as TNF, IL-6, IFN-α2, IFN-γ, IL-1Ra and IL-10. These correlations were observed at day 0 but disappeared at day 28. Gal-9 is released during acute malaria, and reflects its severity. This elevation of Gal-9 in acute malaria infection raises the possibility of its role in termination of the immune response by binding to Tim-3, a receptor of Gal-9.

The factors leading to poor outcomes in malaria infection are incompletely understood. Common genetic variation exists in the human genes for Toll like receptors (TLRs) that alter host responses to pathogen-associated molecular patterns. Genetic variation in TLR1 and TLR6 could alter the risk of development of complicated malaria and ability of the host to control the parasite burden during acute Plasmodium falciparum infection. Five single nucleotide polymorphisms in TLR1 and TLR6 in 432 patients with clinical P. falciparum monoinfection acquired on the Thai-Myanmar border were genotyped. Using logistic regression, associations with the development of complicated malaria and the percentage of infected erythrocytes (parasitaemia) on the day of presentation to clinical care (day zero) were tested. Genotypes carrying the T (major) allele of TLR1 rs5743551—an allele associated with improved outcomes in sepsis—were associated with higher parasitaemia measured on day zero (p = 0.03). Since malaria exerts strong genetic pressure on the human genome, protection from parasitaemia associated with TLR1 rs5743551 may account for the maintenance of an allele associated with poor outcomes in Caucasians with sepsis. These data suggest that genetic variation in TLR1 has effects on the host response to Plasmodium falciparum malaria in Asian populations. Genotypes from TLR6 showed no evidence of association with either complicated malaria or parasite burden.

Knowledge of the population genetics and transmission dynamics of Plasmodium vivax is crucial in predicting the emergence of drug resistance, relapse pattern and novel parasite phenotypes, all of which are relevant to the control of vivax infections. The aim of this study was to analyse changes in the genetic diversity of P. vivax genes from field isolates collected at different times along the Thai–Myanmar border. Two hundred and fifty-four P. vivax isolates collected during two periods 10 years apart along the Thai–Myanmar border were analysed. The parasites were genotyped by nested-PCR and PCR–RFLP targeting selected polymorphic loci of Pvmsp1, Pvmsp3α and Pvcsp genes. The total number of distinguishable allelic variants observed for Pvcsp, Pvmsp1, and Pvmsp3α was 17, 7 and 3, respectively. High genetic diversity was observed for Pvcsp (H E = 0.846) and Pvmsp1 (H E = 0.709). Of the 254 isolates, 4.3 and 14.6 % harboured mixed Pvmsp1 and Pvcsp genotypes with a mean multiplicity of infection (MOI) of 1.06 and 1.15, respectively. The overall frequency of multiple genotypes was 16.9 %. When the frequencies of allelic variants of each gene during the two distinct periods were analysed, significant differences were noted for Pvmsp1 (P = 0.018) and the Pvcsp (P = 0.033) allelic variants. Despite the low malaria transmission levels in Thailand, P. vivax population exhibit a relatively high degree of genetic diversity along the Thai–Myanmar border of Thailand, in particular for Pvmsp1 and Pvcsp, with indication of geographic and temporal variation in frequencies for some variants. These results are of relevance to monitoring the emergence of drug resistance and to the elaboration of measures to control vivax malaria.

Relapse infections resulting from the activation hypnozoites produced by Plasmodium vivax and Plasmodium ovale represent an important obstacle to the successful control of these species. A single licensed drug, primaquine is available to eliminate these liver dormant forms. To date, investigations of vivax relapse infections have been few in number. Genotyping, based on polymorphic regions of two genes (Pvmsp1F3 and Pvcsp) and four microsatellite markers (MS3.27, MS3.502, MS6 and MS8), of 12 paired admission and relapse samples from P. vivax-infected patients were treated with primaquine, revealed that in eight of the parasite populations in the admission and relapse samples were homologous, and heterologous in the remaining four patients. The patients’ CYP2D6 genotypes did not suggest that any were poor metabolisers of primaquine. Parasitaemia tended to be higher in the heterologous as compared to the homologous relapse episodes as was the IgG3 response. For the twelve pro- and anti-inflammatory cytokine levels measured for all samples, only those of IL-6 and IL-10 tended to be higher in patients with heterologous as compared to homologous relapses in both admission and relapse episodes. The data from this limited number of patients with confirmed relapse episodes mirror previous observations of a significant proportion of heterologous parasites in relapses of P. vivax infections in Thailand. Failure of the primaquine treatment that the patients received is unlikely to be due to poor drug metabolism, and could indicate the presence of P. vivax populations in Thailand with poor susceptibility to 8-aminoquinolines.

Ophthalmic safety observations are reported from a clinical trial comparing tafenoquine (TQ) efficacy and safety versus sequential chloroquine (CQ)/primaquine (PQ) for acute Plasmodium vivax malaria.