Detection of Malaria Parasites After Treatment in Travelers: A 12-months Longitudinal Study and Statistical Modelling Analysis

Abstract

The rapid clearance of malaria parasite DNA from circulation has widely been accepted as a fact without being systemically investigated. We assessed the persistence of parasite DNA in travelers treated for Plasmodium falciparum malaria in a malaria-free area. Venous blood was collected at the time of admission and prospectively up to one year. DNA and RNA were extracted and analyzed using species-specific and gametocyte-specific real-time PCR as well as merozoite surface protein 2 (msp2)-PCR. In 31 successfully treated individuals, asexual parasites were seen by microscopy until two days after treatment, whereas parasite DNA was detected by msp2- and species-specific PCR up to days 31 and 42, respectively. Statistical modelling predicted 26% (±0·05 SE) species-specific PCR positivity until day 40 and estimated 48days for all samples to become PCR negative. Gametocytes were detected by microscopy and PCR latest two days after treatment. C_T values correlated well with microscopy-defined parasite densities before but not after treatment started. These results reveal that PCR positivity can persist several weeks after treatment without evidence of viable sexual or asexual parasites, indicating that PCR may overestimate parasite prevalence after treatment.

Keywords: Gametocyte; Malaria; Real-time PCR; Traveler; Treatment; msp2-PCR.

Fig. 1

Prevalence of P. falciparum parasite detected by microscopy and PCR in patients with successful treatment (n = 31). a) Microscopy-defined parasitemias at different time points. Parasitemias declined rapidly during the first two days of treatment initiation. b) Proportion of microscopy positive individuals with available sample at different time-points. Asexual parasites were detected at the most two days after treatment. c) Species-specific qPCR-generated C_T values (presented for all PCR positive samples) over time. qPCR remained positive up to 42 days after treatment. d) Proportion of individuals with species-specific qPCR positive samples over time. All samples collected during days 0–9 are positive by qPCR and thereafter in subsets of samples during different periods. e) Prevalence of infection and number of clones defined by msp2-genotyping PCR. The number of concurrent parasite clones decreased gradually over time. msp2-PCR remained positive the most until day 31 post-treatment. f) Proportion of individuals with positive msp2-PCR at different time points. g) Proportion of gametocyte positive samples by microscopy (gray bars) and gametocyte-specific qPCR (black bars). RNA samples were available for a total of 9 patients. Symbols of the same shape and color (graph a, c, and e) correspond to the same patients in three graphs. Numbers on the bars and over the x-axes (graph b, d, f, and g) represent the total number of individuals with available sample at that time point.

Fig. 2

Local regression fitting of C_T value over time. a) Empty circles represent individual samples at a certain time point and solid red line shows the overall uncertainty which is measured as how well the estimated curve fits the C_T value. b) The solid blue line displays local weighted polynomial regression of C_T value and shaded area shows the 95% confidence interval (CI) [using lm model, followed by loess function in ggplot2].

Fig. 3

Predicted proportion of P. falciparum parasite positive samples by microscopy and species-specific qPCR, over time. The shaded area shows 95% confidence interval (CI). Solid black line shows the model predicted probability of positive microscopy over time. The dotted line represents the model (Cox-HPZ) predicted probability of P. falciparum DNA being detected by species-specific qPCR.