Research Article: Plasmodium falciparum Gametocyte Enrichment in Peripheral Blood Samples by Magnetic Fractionation: Gametocyte Yields and Possibilities to Reuse Columns

Date Published: March 02, 2019

Publisher: The American Society of Tropical Medicine and Hygiene

Author(s): Wouter Graumans, Chiara Andolina, Shehu S. Awandu, Lynn Grignard, Kjerstin Lanke, Teun Bousema.

http://doi.org/10.4269/ajtmh.18-0773

Abstract

Gametocytes are sexual stage malaria parasites responsible for transmission to mosquitoes. Multiple gametocyte-producing clones may be present in natural infections, but the molecular characterization of gametocytes is challenging. Because of their magnetic properties, gametocyte enrichment can be achieved by magnetic fractionation. This increases detection sensitivity and allows specific genotyping of clones that contribute to malaria transmission. Here, we determined the percentage of Plasmodium falciparum gametocytes successfully bound to magnetic activated cell sorting (MACS) LS columns during magnetic fractionation and assessed whether columns can be reused without risking contamination or affecting column binding efficiency. Bound column fractions were quantified using multiplex quantitative reverse transcription polymerase chain reaction (qRT-PCR) for male (pfMGET) and female (CCp4) gametocytes and ring-stage asexual parasites (SBP1). To investigate cross contamination between columns, parasite strain identity was determined by merozoite surface protein 2 genotyping followed by capillary electrophoresis fragment sizing. A reproducible high percentage of gametocytes was bound to MACS LS columns with < 5% gametocytes appearing in the flow-through and < 0.6% asexual ring-stage parasites appearing in the gametocyte fraction. A high yield (> 94%) of gametocyte enrichment was achieved when columns were used up to five times with lower binding success after eight times (79%). We observed no evidence for cross contamination between columns.

Partial Text

Ongoing malaria transmission poses a considerable public health burden in endemic countries worldwide.1 Caused by protozoan parasites of the Plasmodium genus, six species are able to infect human hosts on the bite of infected female Anopheles mosquitoes. Mosquitoes are born uninfected and may become infected after ingestion of blood meals containing circulating mature sexual stages of the parasite, known as gametocytes. Once ingested by the mosquito, gametocytes fertilize and, following sporogonic development, produce hundreds to thousands sporozoites that migrate to the salivary glands of the mosquito and render the mosquito infectious to humans on their next bite.

The aims of this study were to determine the proportion of P. falciparum gametocytes successfully bound to MACS LS columns during magnetic fractionation and to assess whether columns can be reused without risking contamination or affecting column binding efficiency.

 

Source:

http://doi.org/10.4269/ajtmh.18-0773

 

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