Comparison of molecular quantification of Plasmodium falciparum gametocytes by Pfs25 qRT-PCR and QT-NASBA in relation to mosquito infectivity

Abstract

BackgroundQuantifying gametocyte densities in natural malaria infections is important to estimate malaria transmission potential. Two molecular methods (Pfs25 mRNA quantitative reverse transcriptase PCR (qRT-PCR) and Pfs25 mRNA quantitative nucleic acid sequence based amplification (QT-NASBA)) are commonly used to determine gametocyte densities in clinical and epidemiological studies and allow gametocyte detection at densities below the microscopic threshold for detection. Here, reproducibility of these measurements and the association between estimated gametocyte densities and mosquito infection rates were compared.MethodsTo quantify intra- and inter-assay variation of QT-NASBA and qRT-PCR, a series of experiments was performed using culture-derived mature Plasmodium falciparum gametocytes from three different parasite isolates (NF54, NF135, NF166). Pfs25 mRNA levels were also determined in samples from clinical trials in Mali and Burkina Faso using both methods. Agreement between the two methods and association with mosquito infection rates in membrane feeding assays were assessed.ResultsIntra- and inter-assay variability was larger in QT-NASBA compared to qRT-PCR, particularly at low gametocyte densities (< 1 gametocyte per μL). Logistic models, including log-transformed gametocytaemia estimated by QT-NASBA, explained variability in mosquito feeding experiment results as well as log-transformed gametocytaemia by qRT-PCR (marginal R2 0.28 and 0.22, respectively). Densities determined by both methods strongly correlated with mosquito infection rates [Spearman’s rank correlation coefficient, 0.59 for qRT-PCR and 0.64 for QT-NASBA (P < 0.001 for both)]. Gametocyte densities estimated by qRT-PCR were higher than levels estimated by QT-NASBA or light microscopy at high densities (>100 gametocyte per μL). Samples collected in one of the two transmission studies had extremely low gametocyte densities by both molecular methods, which is suggestive of RNA degradation due to an unknown number of freeze–thaw cycles and illustrates the reliance of molecular gametocyte diagnostics on a reliable cold-chain.ConclusionsThe experiments indicate that both qRT-PCR and QT-NASBA are of value for quantifying mature gametocytes in samples collected in field studies. For both assays, estimated gametocyte densities correlated well with mosquito infection rates. QT-NASBA is less reproducible than qRT-PCR, particularly for low gametocyte densities.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1584-z) contains supplementary material, which is available to authorized users.

Molecular Markers for Sensitive Detection of Plasmodium falciparum Asexual Stage Parasites and their Application in a Malaria Clinical Trial

“…An important caveat is that our markers were selected based on expression in a single laboratory-adapted parasite line, NF54. Although we previously assessed SBP1 transcript levels in 3D7 4 and Pfs25 transcript levels in NF54, NF135, and NF166 parasites 38 and found highly comparable levels between parasite isolates, this does not mean that in vitro expression can be directly extrapolated to expression in in vivo samples. Our quantitative approach may thus be affected by possible differences between in vitro-cultured parasites and patient samples.…”

RETRACTED ARTICLE: Molecular detection and quantification of Plasmodium falciparum gametocytes carriage in used RDTs in malaria elimination settings in northern Senegal

“…Since mosquito infection requires the presence of gametocytes in humans, knowledge of the epidemiology of gametocyte carriage is an essential parameter for assessing malaria transmission and predicting infectiousness in vectors [1,7,8]. Some molecular techniques are used today for the detection and quantification of gametocytes [1,9]. The complexity and cost of these tests limits their use in low resource areas.…”

Assessing Malaria Vaccine Efficacy

“…Moreover, it seems that infected mosquitoes can cluster in discrete locations [105], thus requiring extensive studies on the distribution of infected mosquitos before and after vaccine trials. Since the number of infected mosquitoes depend on the number of circulating gametocytes [106], defining the number of gametocyte carrier prior to vaccine implementation is also a pre-requisite. In addition, defining transmission intensity of the vaccine site is important since it may influence the outcome of the vaccination.…”