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01.12.2012 | Research | Ausgabe 1/2012 Open Access

Malaria Journal 1/2012

Quantification of multiple infections of Plasmodium falciparum in vitro

Zeitschrift:
Malaria Journal > Ausgabe 1/2012
Autoren:
Mark A Wacker, Lindsey B Turnbull, Leah A Walker, Michael C Mount, Michael T Ferdig
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1475-2875-11-180) contains supplementary material, which is available to authorized users.

Competing interest

The authors declare that they have no competing interests.

Authors’ contributions

MAW and MTF conceived and designed the experiments. MAW, LBT, LAW and MCM performed the experiments and analysed the data. MAW, LBT and MTF wrote the manuscript. All authors read and approved the final manuscript.

Abstract

Background

Human malaria infections caused by the parasite Plasmodium falciparum often contain more than one genetically distinct parasite. Despite this fact, nearly all studies of multiple strain P. falciparum infections have been limited to determining relative densities of each parasite within an infection. In light of this, new methods are needed that can quantify the absolute number of parasites within a single infection.

Methods

A quantitative PCR (qPCR) method was developed to track the dynamic interaction of P. falciparum infections containing genetically distinct parasite clones in cultured red blood cells. Allele-specific primers were used to generate a standard curve and to quantify the absolute concentration of parasite DNA within multi-clonal infections. Effects on dynamic growth relationships between parasites under drug pressure were examined by treating mixed cultures of drug sensitive and drug resistant parasites with the anti-malarial drug chloroquine at different dosing schedules.

Results

An absolute quantification method was developed to monitor the dynamics of P. falciparum cultures in vitro. This method allowed for the observation of competitive suppression, the reduction of parasites numbers due to the presence of another parasite, and competitive release, the improved performance of a parasite after the removal of a competitor. These studies demonstrated that the presence of two parasites led to the reduction in density of at least one parasite. The introduction of drug to a mixed culture containing both a drug resistant and drug sensitive parasites resulted in an increased proportion of the drug resistant parasite. Moreover, following drug treatment, the resistant parasite experienced competitive release by exhibiting a fitness benefit greater than simply surviving drug treatment, due to the removal of competitive suppression by the sensitive parasite.

Conclusions

The newly developed assay allowed for the examination of the dynamics of two distinct clones in vitro; both competitive suppression and release were observed. A deeper understanding of the dynamic growth responses of multiple strain P. falciparum infections, with and without drug pressure, can improve the understanding of the role of parasite interactions in the spread of drug resistant parasites, perhaps suggesting different treatment strategies.
Zusatzmaterial
Additional file 1: Standard curves of all clones A) Dd2 DNA alone (black circles) produces clear standard curve (R 2  = 0.999) and adding HB3 DNA (red squares) does not alter this curve (R 2  = 0.998). B) HB3 DNA alone (black circles) produces a clear standard curve (R2 = 0.999) and neither the addition of Dd2 DNA (red squares) nor 7C424 DNA (blue diamonds) significantly alters the curve (R2 = 0.999, and 0.932 respectively). C) 7C424 DNA alone (blue diamonds) produces a clear standard curve (R2 = 0.999) and adding HB3 DNA (black squares) does not significantly alter the curve (R 2  = 0.997). (JPEG 939 KB)
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Authors’ original file for figure 1
12936_2011_2438_MOESM2_ESM.pdf
Authors’ original file for figure 2
12936_2011_2438_MOESM3_ESM.pdf
Authors’ original file for figure 3
12936_2011_2438_MOESM4_ESM.pdf
Literatur
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