Recrudescence, Reinfection, or Relapse? A More Rigorous Framework to Assess Chloroquine Efficacy for Plasmodium vivax Malaria

Abstract

Background: Plasmodium vivax resistance to chloroquine (CQ) has been reported worldwide, although the World Health Organization clinical drug efficacy studies protocol does not permit classification of patient outcomes.

Methods: We enrolled 40 patients with P. vivax malaria in northeastern Cambodia, where >17% treatment failures were previously reported. Patients were treated with CQ (30 mg/kg) and followed for 2 months, with frequent clinical examination and capillary blood sample collection for microscopy, molecular parasite detection and genotyping, and drug concentration measurements. Reinfections were prevented by relocating patients to a transmission-free area.

Results: P. vivax parasites were eliminated in all patients by day 3. Genomic analyses revealed that all clones in polyclonal infections were cleared at the same rate, indicating their equal susceptibility to CQ. CQ blood concentrations were below the therapeutic level in all recurrent infections (24 of 40 patients), which were efficiently cleared by a second course of CQ treatment. Genotyping (128 SNPs barcode) and sequences of entire parasite genome (Whole-Genome Sequencing, Illumina) indicated that two thirds (6 of 8) of the recurrent parasites resulted from heterologous relapses whose 50% are from by sibling/recombinant clones.

Conclusions: No evidence of CQ resistance was observed. Our data suggest that P. vivax antimalarial drug resistance is likely overestimated and that the current guidelines for clinical drug studies of P. vivax malaria need to be revised.

Figure 1.

Relative proportion of clones in polyclonal infections upon a 3-day supervised regimen of chloroquine (30 mg/kg), Ratanakiri, Cambodia, 2014. Allele frequencies (AFs) for 2 patients with complex infections (patient BL12 [A] and patient BL13 [B]) at each single-nucleotide polymorphism (black dots) before (x-axis) and 8 hours after (y-axis) treatment. See also Supplementary Table 2.

Figure 2.

Cumulative proportion of patients with nonrecrudescent infection detected by real-time polymerase chain reaction, Ratanakiri, Cambodia, 2014. Patients were or were not relocated to a malaria-free area and were treated with a 3-day supervised regimen of chloroquine (30 mg/kg). The ratio of the hazards for recrudescence for nonrelocated versus relocated patients was 0.86 (95% confidence interval [CI], .38–1.92; P = .7 by the log-rank test); The median time to recurrence (in days) was similar between nonrelocated patients (49.0; 95% CI, 49.0–56.0) and relocated patients (49.0; 95% CI, 43.0–59.0).

Figure 3.

Whole-blood drug concentrations in Plasmodium vivax–infected patients receiving a 3-day supervised regimen of chloroquine (CQ; 30 mg/kg), Ratanakiri, Cambodia, 2014. Data are expressed as the sum of the concentrations of CQ and N-desethyl chloroquine (DCQ). A, Concentrations on day 7 for patients with and those without recurrences during the monitoring period. B, CQ blood concentrations on day 28 and the day of recurrence (detected by real-time polymerase chain reaction [PCR]) in patients experiencing recurrences during the 2-month monitoring period. C, Mean pharmacokinetic profile (±SD) over time (line) and cumulative proportion of parasite recurrences detected by real-time PCR (histograms). In panels A and B, CQ blood concentrations above the threshold for resistance (ie, 100 ng/mL) are in red, concentrations from 70 to 100 ng/mL are in orange, and concentrations <70 ng/mL are in green.