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A multiplex assay for the sensitive detection and quantification of male and female Plasmodium falciparum gametocytes

Meerstein-Kessel, Lisette and Andolina, Chiara and Carrio, Elvira and Mahamar, Almahamoudou and Sawa, Patrick and Diawara, Halimatou and van de Vegte-Bolmer, Marga and Stone, Will and Collins, Katharine A. and Schneider, Petra and Dicko, Alassane and Drakeley, Chris and Felger, Ingrid and Voss, Till and Lanke, Kjerstin and Bousema, Teun. (2018) A multiplex assay for the sensitive detection and quantification of male and female Plasmodium falciparum gametocytes. Malaria journal, 17 (1). p. 441.

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Abstract

The transmission of malaria to mosquitoes depends on the presence of gametocytes that circulate in the peripheral blood of infected human hosts. Sensitive estimates of the densities of female gametocytes (FG) and male gametocytes (MG) may allow the prediction of infectivity to mosquitoes and thus a molecular estimate of the human infectious reservoir for transmission.; A novel multiplex qRT-PCR assay with intron-spanning primers was developed for the parallel quantification of FG and MG. CCp4 (PF3D7_0903800) transcripts specific for FG and PfMGET (PF3D7_1469900) transcripts specific for MG were quantified in total nucleic acids. The assay was validated on sex-sorted gametocytes from culture material and on samples from clinical trials with gametocytocidal drugs. Synthetic RNA standards were generated for the two targets genes and calibrated against known gametocyte quantities.; The limit of detection was determined at 0.1 male and 0.1 female gametocyte/µL, which was equal to the limit of quantification (LOQ) for MG, while the LOQ for FG was 1 FG/µL. Results from previously reported clinical trials that used separate gametocyte qRT-PCR assays for FG (targeting Pfs25) and MG (targeting PfMGET) were reproduced with the multiplex assay. High levels of agreement between separate assays and the multiplex approach were observed (R; 2; = 0.9473, 95% CI 0.9314-0.9632, for FG measured by transcript levels of Pfs25 in qRT-PCR or CCp4 in multiplex; R; 2; = 0.8869, 95% CI 0.8541-0.9197, for MG measured by PfMGET in either single or multiplex qRT-PCR). FG and MG transcripts were detected in pure ring stage parasites at 10,000- and 100,000-fold reduced frequency for CCp4 and PfMGET, respectively. The CCp4 and PfMGET transcripts were equally stable under suboptimal storage conditions.; Gametocyte densities and their sex ratios can be determined in the presented one-step multiplex assay with higher throughput than single assays. The interpretation of low gametocyte densities at asexual parasite densities above 1000 parasites/µL requires caution to avoid false positive gametocyte signals from spurious transcript levels in ring stage parasites.
Faculties and Departments:09 Associated Institutions > Swiss Tropical and Public Health Institute (Swiss TPH)
09 Associated Institutions > Swiss Tropical and Public Health Institute (Swiss TPH) > Department of Medical Parasitology and Infection Biology > Gene Regulation (Voss)
09 Associated Institutions > Swiss Tropical and Public Health Institute (Swiss TPH) > Department of Medical Parasitology and Infection Biology > Molecular Diagnostics (Felger)
UniBasel Contributors:Carrio Gaspar, Elvira and Felger, Ingrid and Voss, Till
Item Type:Article, refereed
Article Subtype:Research Article
Publisher:BioMed Central
ISSN:1475-2875
Note:Publication type according to Uni Basel Research Database: Journal article
Language:English
Identification Number:
Last Modified:11 Dec 2018 10:18
Deposited On:11 Dec 2018 10:18

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