Quantification of female and male Plasmodium falciparum gametocytes by reverse transcriptase quantitative PCR

Petra Schneider,Sarah E Reece,Ben C.L Van Schaijk,Teun Bousema,Kjerstin H.W Lanke,Cora S.J Meaden,Amal Gadalla,Lisa C Ranford-Cartwright,Hamza A Babiker

doi:10.1016/j.molbiopara.2015.03.006

Abstract

The transmission of malaria parasites depends on the presence of sexual stages (gametocytes) in the blood, making the ratio and densities of female and male gametocytes important determinants of parasite fitness. This manuscript describes the development of reverse transcriptase quantitative PCR (RT-qPCR) assays to separately quantify mature female and male gametocytes of the human malaria parasite Plasmodium falciparum, and reveals that Pfs25 mRNA is expressed only in female gametocytes. The female (Pfs25) and male (Pfs230p) gametocyte specific RT-qPCR assays have lower detection limits of 0.3 female and 1.8 male gametocytes per microlitre of blood, respectively, making them more sensitive than microscopy. Accurate quantification of the ratio and densities of female and male gametocytes will increase understanding of P. falciparum transmission and improve the evaluation of transmission blocking interventions.

Highlights

The transmission of malaria parasites depends on the presence of sexual stages in the blood, making the ratio and densities of female and male gametocytes important determinants of parasite fitness
This manuscript describes the development of reverse transcriptase quantitative PCR (RT-qPCR) assays to separately quantify mature female and male gametocytes of the human malaria parasite Plasmodium falciparum, and reveals that Pfs25 mRNA is expressed only in female gametocytes
When the proportion of female gametocytes in each sample (0.36%, 95% and 98% for the “Purified males”, “Purified females”, “Mixed sex gametocytes” 3D7-1 and 3D7-2, respectively) is taken into account, there are no significant differences in Pfs25 mRNA expression per female gametocyte across these sex ratios (F3,178 = 2.63, p = 0.052; Fig. 2A)

Summary

Introduction

This manuscript describes the development of reverse transcriptase quantitative PCR (RT-qPCR) assays to separately quantify mature female and male gametocytes of the human malaria parasite Plasmodium falciparum, and reveals that Pfs25 mRNA is expressed only in female gametocytes. The female (Pfs25) and male (Pfs230p) gametocyte specific RT-qPCR assays have lower detection limits of 0.3 female and 1.8 male gametocytes per microlitre of blood, respectively, making them more sensitive than microscopy.

Results

Conclusion