Image: Blood smear showing a male gametocyte of Plasmodium vivax (Photo courtesy of Dr. Mae Melvin).
Image: Magnetic-activated cell sorting (MACS) equipment (Photo courtesy the University of South Carolina).
Gametocytes are the transmission stages of malaria parasites and as their density in the human host is typically low, they are often undetected by conventional light microscopy.
A detailed comparison of three methods, namely light microscopy, magnetic fractionation (MF) and reverse transcriptase polymerase chain reaction (RT-PCR) for detection of Plasmodium falciparum
and P. vivax
gametocytes has been conducted.
Scientists at the University of Western Australia (Fremantle, Australia) working with an international team took blood samples from total of 70 children, 56 with P. falciparum
and 14 with P. vivax
and aged between six months and five years. For the light microscopy Giemsa-stained thick blood smears were examined and parasite density quantified independently by two skilled microscopists with parasites counted against 500 white cells.
For the MF study the samples were subjected to high field gradient MF and magnetic particles (Spherotech; Lake Forest, IL, USA) were added to achieve a total known particle concentration of 100 particles per μL of blood. The samples were then magnetically fractionated using magnetic-activated cell sorting (MACS) equipment (Miltenyi Biotec; Bergisch Gladbach, Germany). For the RT-PCR study the team used the P. falciparum
Pfs25 gene or the P. vivax
Pvs25 gene copy number as a measure for gametocyte abundance. All RT-PCR assays were run in 384-well plate format on the LightCycler480 platform (Roche; Basel, Switzerland).
The scientists found that MF and RT-PCR were similarly sensitive and specific, and both were superior to light microscopy (LM). Overall, there were approximately 20% gametocyte-positive samples by LM, whereas gametocyte positivity by MF and RT-PCR were both more than two-fold this level. In the subset of samples collected prior to treatment, 29% of children were positive by LM, and 85% were gametocyte positive by MF and RT-PCR, respectively. MF is a feasible methodology for field-based gametocyte detection that can be applied even in larger scale studies. With an appropriate set- up, four to six samples could conveniently be processed by one person in an hour with results being available for all samples after approximately 90 minutes.
The authors concluded that that magnetic fractionation is a very good alternative method for on-site gametocyte detection under field conditions. The method has, as previously shown under laboratory conditions, similar sensitivity to RT-PCR and can be used to derive quantitative gametocyte densities for both P. falciparum
and P. vivax
. The study was published on August 14, 2014, in the Malaria Journal.
University of Western Australia