Examining Plasmodium falciparum and P. vivax clearance subsequent to antimalarial drug treatment in the Myanmar-China border area based on quantitative real-time polymerase chain reaction

Abstract

Background: Recent emergence of artemisinin-resistant P. falciparum has posed a serious hindrance to the elimination of malaria in the Greater Mekong Subregion. Parasite clearance time, a measure of change in peripheral parasitaemia in a sequence of samples taken after treatment, can be used to reflect the susceptibility of parasites or the efficiency of antimalarials. The association of genetic polymorphisms and artemisinin resistance has been documented. This study aims to examine clearance time of P. falciparum and P. vivax parasitemia as well as putative gene mutations associated with residual or recurred parasitemia in Myanmar.

Methods: A total of 63 P. falciparum and 130 P. vivax samples collected from two internally-displaced populations and one surrounding village were examined for parasitemia changes. At least four samples were taken from each patient, at the first day of diagnosis up to 3 months following the initial treatment. The amount of parasite gene copy number was estimated using quantitative real-time PCR based on a species-specific region of the 18S rRNA gene. For samples that showed residual or recurred parasitemia after treatment, microsatellites were used to identify the 'post-treatment' parasite genotype and compared such with the 'pre-treatment' genotype. Mutations in genes pfcrt, pfmdr1, pfatp6, pfmrp1 and pfK13 that are potentially associated with ACT resistance were examined to identify if mutation is a factor for residual or persistent parasitemia.

Results: Over 30% of the P. falciprium infections showed delayed clearance of parasitemia after 2-3 days of treatment and 9.5% showed recurred parasitemia. Mutations in codon 876 of the pfmrp1 corroborated significance association with slow clearance time. However, no association was observed in the variation in pfmdr1 gene copy number as well as mutations of various codonsinpfatp6, pfcrt, and pfK13 with clearance time. For P. vivax, over 95% of the infections indicated cleared parasitemia at days 2-3 of treatment. Four samples were found to be re-infected with new parasite strains based on microsatellite genotypes after initial treatment.

Conclusion: The appearance of P.falciparum infected samples showing delayed clearance or recurred parasitemia after treatment raises concerns on current treatment and ACT drug resistance.

Keywords: Artemisinin-combined therapy; Malaria; Microsatellite; P. falciparum; P. vivax; Parasite clearance; Quantitative PCR; Resistance genes.

Fig. 1

Change in Plasmodium vivax parasitemia among follow-up samples collected from day-0 (the day when the patient was admitted to the hospital and sought antimalarial treatment) to up to 6-months after treatment. Samples with cleared parasitemia at day-2 a, day-3 b, at day-7 and after c, and with recurred parasitemia d were presented. For the samples that showed recurred infection, the initial and recurred genotypes were found to be different based on microsatellites, suggestive of a newly infected parasite strain that caused recurred parasitemia after initial drug treatment

Fig. 2

Change in Plasmodium falciparum parasitemia among follow-up samples collected from day-0 (the day when the patient was admitted to the hospital and sought antimalarial treatment) to up to 6-months after treatment. Samples with cleared parasitemia at day-2 a, day-3 b, at day-7 and after c, and with recurred parasitemia d were presented. For the samples that showed recurred infection, two samples showed identical microsatellite genotypes

Fig. 3

Boxplot comparing initial parasitemia of P. falciparum samples that indicated fast (gray box; parasite cleared at day 2 or 3) and delayed (white box; parasite cleared after day 3) parasite clearance between two age groups (aged below and under 18). Number above bar indicates number of samples included. Asterisk indicates level of significance

Fig. 4

Scatter plot based on Principal Component Analysis (PCA) of microsatellite data among the fast (purple color; no parasite detected at day-3) and delayed (yellow color; parasite detected after day-3) clearance samples of P. falciparum

Fig. 5

Frequency of mutations in various gene codons among samples that showed fast (parasite cleared at day 2 or 3) and delayed (parasite cleared after day 3) clearance of P. falciparum. Bold denotes codon of which the mutation frequency is significantly associated with the parasite clearance time