Association between in vivo and ex vivo Plasmodium vivax resistance to chloroquine in Brazilian Amazon isolates

Background: One of the major obstacles to malaria control and elimination efforts is the spread of drug-resistant parasites. Some studies in Brazilian Amazon estimated 5-10% resistance of Plasmodium vivax to chloroquine (CQ) in vivo and 9-11% ex vivo. However, there are no studies showing an association between resistance in vivo and ex vivo. The objective of this study is to associate resistance to CQ in vivo and ex vivo in isolates of P. vivax from the Brazilian Amazon. Materials and Methods: Were included P. vivax isolates from patients diagnosed at Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (Manaus, Amazonas - Brazil), previously to CQ and primaquine treatment, followed-up for 42 days. Were obtained samples to make thick blood smear and the microtest test for ex vivo drug susceptibility. All patients presenting new malaria episode until D42 and CQ plasmatic levels >100nM during recurrence (DR) were considered as CQ-resistants in vivo. Isolates presenting IC50% (inhibitory concentration 50%) ≥100 nM were considered ex vivo resistants. Samples with parasitemia ≥2 crosses (501.0-10,000.0 parasites/mm3) and 60% of ring stages vivax parasites were cultivated in presence of CQ diphosphate (1.95-1000 nM), during 42 hours and then were realized the schizonts counting by light microscopy (LM) and the IC50% calculation. Results: Were included 262 patients in this study. Three (1.2%) presented recurrence until D42 visit, however they were not considered in vivo resistant because their CQ plasmatic levels were <100 nM at DR. A total of 7.3% of the total isolates (19/262) were selected to microtest and showed parasitemia mean of 6,277.0 (CI95%: 3,653.3-8,900.7). Fourteen isolates were analysed by LM and 2 were considered with ex vivo resistance. The mean IC50% from ex vivo resistant isolates (2/14) was 271.5 nM (CI95%: 190.4-352.6) and 6.8 nM (CI95%: 1.5-12.2) from the sensitive ones (12/14) (p<0.001, unpaired t test). Conclusions: The frequence of CQ-resistant isolates and the IC50% values of CQ-sensitives were lower than the previously found in this region. Our results indicate a decrease of P. vivax resistance to CQ and a weak association between P. vivax resistance in vivo and ex vivo.