Evaluation of Plasmodium falciparum gametocyte detection in different patient material

Katharina Kast,Teferi Eshetu,Andreas Wieser,Jonathan Shock,Ahmed Zeynudin,Nuredin Abduselam,Nicole Berens-Riha,Michael Pritsch,Thomas Löscher

doi:10.1186/1475-2875-12-438

Abstract

BackgroundFor future eradication strategies of malaria it is important to control the transmission of gametocytes from humans to the anopheline vector which causes the spread of the disease. Sensitive, non-invasive methods to detect gametocytes under field conditions can play a role in monitoring transmission potential.MethodsMicroscopically Plasmodium falciparum-positive patients from Jimma, Ethiopia donated finger-prick blood, venous blood, saliva, oral mucosa and urine samples that were spotted on filter paper or swabs. All samples were taken and stored under equal, standardized conditions. RNA was extracted from the filter paper and detected by real-time QT-NASBA. Pfs16-mRNA and Pfs25-mRNA were measured with a time to positivity to detect gametocyte specific mRNA in different gametocyte stages. They were compared to 18S-rRNA, which is expressed in all parasite stages. Results were quantified via a known dilution series of artificial RNA copies.ResultsNinety-six samples of 16 uncomplicated malaria patients were investigated. 10 (66.7%) of the slides showed gametocyte densities between 0.3-2.9 gametocytes/μl. For all RNA-targets, molecular detection in blood samples was most sensitive; finger-prick sampling required significantly smaller amounts of blood than venous blood collection. Detection of asexual 18S-rRNA in saliva and urine showed sensitivities of 80 and 67%, respectively. Non-invasive methods to count gametocytes proved insensitive. Pfs16-mRNA was detectable in 20% of urine samples, sensitivities for other materials were lower. Pfs25-mRNA was not detectable in any sample.ConclusionsThe sensitivity of non-invasively collected material such as urine, saliva or mucosa seems unsuitable for the detection of gametocyte-specific mRNA. Sensitivity in asymptomatic carriers might be generally even lower. Finger-prick testing revealed the highest absolute count of RNA copies per μL, especially for Pfs25-mRNA copies. The method proved to be the most effective and should preferably be applied in future transmission control and eradication plans. A rapid test for gametocyte targets would simplify efforts.

Highlights

For future eradication strategies of malaria it is important to control the transmission of gametocytes from humans to the anopheline vector which causes the spread of the disease
The presence of mature Plasmodium falciparum sexually committed parasites, so-called stage V gametocytes, is necessary for transmission from humans to anopheline vectors, while the asexual stages are responsible for the clinical symptoms of the disease
It was previously shown that gametocyte densities below the detection limit of microscopy can still lead to mosquito infection [3,4,5,6]

Summary

Introduction

For future eradication strategies of malaria it is important to control the transmission of gametocytes from humans to the anopheline vector which causes the spread of the disease. The presence of mature Plasmodium falciparum sexually committed parasites, so-called stage V gametocytes, is necessary for transmission from humans to anopheline vectors, while the asexual stages are responsible for the clinical symptoms of the disease. It was previously shown that gametocyte densities below the detection limit of microscopy can still lead to mosquito infection [3,4,5,6]. To bridge this gap and to quantify the infectious reservoir, modern molecular techniques were introduced [7,8]. Quantitative nucleic acid sequence based amplification (QT-NASBA) was used, which offers a limit of detection (LOD) down to 0.02 gametocytes per μL [9]

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Results

Discussion

Conclusion