Plasmodium falciparum population dynamics during the early phase of anti-malarial drug treatment in Tanzanian children with acute uncomplicated malaria

Abstract

Background: This study aimed to explore Plasmodium falciparum population dynamics during the early phase of anti-malarial drug treatment with artemisinin-based combination therapy in children with clinical malaria in a high transmission area in Africa.

Methods: A total of 50 children aged 1-10 years with acute uncomplicated P. falciparum malaria in Bagamoyo District, Tanzania, were enrolled. Participants were hospitalized and received supervised standard treatment with artemether-lumefantrine according to body weight in six doses over 3 days. Blood samples were collected 11 times, i.e. at time of diagnosis (-2 h) and 0, 2, 4, 8, 16, 24, 36, 48, 60 and 72 h after initiation of treatment. Parasite population dynamics were assessed using nested polymerase chain reaction (PCR)-genotyping of merozoite surface protein (msp) 1 and 2.

Results: PCR-analyses from nine sequential blood samples collected after initiation of treatment identified 20 and 21 additional genotypes in 15/50 (30%) and 14/50 (28%) children with msp1 and msp2, respectively, non-detectable in the pre-treatment samples (-2 and 0 h combined). Some 15/20 (75%) and 14/21 (67%) of these genotypes were identified within 24 h, whereas 17/20 (85%) and 19/21 (90%) within 48 h for msp1 and msp2, respectively. The genotype profile was diverse, and varied considerably over time both within and between patients, molecular markers and their respective families.

Conclusion: PCR analyses from multiple blood samples collected during the early treatment phase revealed a complex picture of parasite sub-populations. This underlines the importance of interpreting PCR-outcomes with caution and suggests that the present use of PCR-adjustment from paired blood samples in anti-malarial drug trials may overestimate assessment of drug efficacy in high transmission areas in Africa.The study is registered at http://www.clinicaltrials.gov with identifier NCT00336375.

Figure 1

P. falciparum clearance measured as proportion of patients with positive blood slides (asexual parasitaemia) and PCR results (defined as a positive amplification in at least one genetic marker, i.e. msp1 or msp2) at each blood sampling point during the study. (Error bars represent 95% confidence intervals)

Figure 2

Overall distribution and frequencies of individual genotypes during follow-up in the entire cohort. Individual genotypes are represented on the x-axis with the frequency at which they occur (left y-axis) by each blood sampling point during the study (right y-axis)

Figure 3

Accumulated proportion of primary identification during follow-up of msp1 (N = 20, solid line) and msp2 (N = 21, broken line) genotypes exclusively identified after initiation of treatment

Figure 4

Distribution of detectable msp1genotypes over time for each study participant

Figure 5

Distribution of detectable msp2genotypes over time for each study participant

Figure 6

Example of different PCR patterns. Patient ID 2 is an example of intermittent genotype pattern, whereas ID 29 shows continuous fading pattern in all markers and ID 48 a continuous fading pattern with random bands in msp2 IC and a random pattern in msp1 MAD20. *Patient ID 2 is followed by a positive control for msp2 (FC27) and msp1 (R033)