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A multiplex assay for the sensitive detection and quantification of male and female Plasmodium falciparum gametocytes.
Meerstein-Kessel, Lisette; Andolina, Chiara; Carrio, Elvira; Mahamar, Almahamoudou; Sawa, Patrick; Diawara, Halimatou; van de Vegte-Bolmer, Marga; Stone, Will; Collins, Katharine A; Schneider, Petra; Dicko, Alassane; Drakeley, Chris; Felger, Ingrid; Voss, Till; Lanke, Kjerstin; Bousema, Teun.
Afiliación
  • Meerstein-Kessel L; Department of Medical Microbiology, Radboud University Medical Center, Nijmegen, The Netherlands.
  • Andolina C; Centre for Molecular and Biomolecular Informatics, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands.
  • Carrio E; Department of Medical Microbiology, Radboud University Medical Center, Nijmegen, The Netherlands.
  • Mahamar A; Swiss Tropical and Public Health Institute, Basel, Switzerland.
  • Sawa P; University of Basel, Basel, Switzerland.
  • Diawara H; Malaria Research and Training Centre, University of Science, Techniques and Technologies of Bamako, Bamako, Mali.
  • van de Vegte-Bolmer M; Human Health Division, International Centre for Insect Physiology and Ecology, Mbita Point, Kenya.
  • Stone W; Malaria Research and Training Centre, University of Science, Techniques and Technologies of Bamako, Bamako, Mali.
  • Collins KA; Department of Medical Microbiology, Radboud University Medical Center, Nijmegen, The Netherlands.
  • Schneider P; Department of Immunology and Infection, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK.
  • Dicko A; Department of Medical Microbiology, Radboud University Medical Center, Nijmegen, The Netherlands.
  • Drakeley C; Institute of Evolutionary Biology and Institute of Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, Edinburgh, UK.
  • Felger I; Malaria Research and Training Centre, University of Science, Techniques and Technologies of Bamako, Bamako, Mali.
  • Voss T; Department of Immunology and Infection, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK.
  • Lanke K; Swiss Tropical and Public Health Institute, Basel, Switzerland.
  • Bousema T; Swiss Tropical and Public Health Institute, Basel, Switzerland.
Malar J ; 17(1): 441, 2018 Nov 29.
Article en En | MEDLINE | ID: mdl-30497508
BACKGROUND: 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. METHODS: 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. RESULTS: 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 (R2 = 0.9473, 95% CI 0.9314-0.9632, for FG measured by transcript levels of Pfs25 in qRT-PCR or CCp4 in multiplex; R2 = 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. CONCLUSIONS: 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.
Asunto(s)

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Plasmodium falciparum / Malaria Falciparum / Parasitemia / Técnicas de Diagnóstico Molecular / Carga de Parásitos / Reacción en Cadena en Tiempo Real de la Polimerasa Tipo de estudio: Diagnostic_studies / Guideline Límite: Female / Humans / Male Idioma: En Revista: Malar J Asunto de la revista: MEDICINA TROPICAL Año: 2018 Tipo del documento: Article País de afiliación: Países Bajos Pais de publicación: Reino Unido

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Plasmodium falciparum / Malaria Falciparum / Parasitemia / Técnicas de Diagnóstico Molecular / Carga de Parásitos / Reacción en Cadena en Tiempo Real de la Polimerasa Tipo de estudio: Diagnostic_studies / Guideline Límite: Female / Humans / Male Idioma: En Revista: Malar J Asunto de la revista: MEDICINA TROPICAL Año: 2018 Tipo del documento: Article País de afiliación: Países Bajos Pais de publicación: Reino Unido