The recent focus on the elimination of malaria has led to an increased interest in the role of sexual stages in its transmission. We introduce Plasmodium falciparum gametocyte exported protein-5 PfGEXP5 transcript analysis as an important tool for evaluating the earliest ring stage sexual gametocytes in the blood of infected individuals.
We Parasitemia asexual reproduction that gametocyte rings are detected in the peripheral blood immediately following establishment of asexual infections—without the need for triggers such as high-density asexual "Parasitemia asexual reproduction" or drug treatment.
Committed gametocytes are refractory to the commonly used drug piperaquine, and mature gametocytes reappear in the bloodstream 10 days after the initial appearance of gametocyte rings.
A further wave of commitment is observed following recrudescent asexual parasitemia, and these gametocytes are again refractory to piperaquine treatment. This work has implications for monitoring gametocyte and transmission dynamics and responses to drug treatment. Treatment of malaria is heavily reliant on a class of drugs called the artemisinins, delivered as a combination with a limited repertoire of partner drugs. Resistance to the partner drug piperaquine is a particular emerging problem in Cambodia, underpinning lower cure rates [ 4—6 ].
As the World Health Organization WHO shifts its focus from disease control to elimination, it is critically important to understand the drivers and dynamics of carriage of both asexual parasites, which cause disease, and sexual-stage gametocytes, which are responsible for disease transmission.
Gametocytogenesis is initiated when a sexually committed merozoite invades a red blood cell RBC [ 7 ]. The sexually committed parasite remains inside its host RBC but undergoes a remarkable morphological change as it transforms from a cell optimized for multiplication in the bloodstream of humans to a cell capable of undergoing sexual reproduction in a mosquito.
Early Parasitemia asexual reproduction stage gametocytes are morphologically indistinguishable from asexual rings. Stage II—IV gametocytes disappear from the circulation, apparently by sequestering in deep tissues, including the spleen and bone marrow [ 9 ].
The only morphologically recognizable gametocyte stage observed in the peripheral circulation in humans is stage V, which re-enters the peripheral circulation and becomes available for uptake by mosquitoes. Given the lack of specific markers for ring-stage gametocytes, it has been difficult to answer a number "Parasitemia asexual reproduction" fundamental questions, such as whether these early stage gametocytes are sequestered or are freely circulating [ 910 ].
Indeed, questions remain as to whether gametocyte commitment occurs in the bloodstream or in a privileged environment such as the bone marrow. Similarly, it is not clear whether gametocyte production is a constitutive event, with a subpopulation of parasites converting Parasitemia asexual reproduction sexual development during each asexual replication cycle, or an event, triggered by exposure to density-dependent changes in nutrient conditions or by environmental stresses, such as a host immune response [ 11—14 ].
Importantly, it has been suggested that exposure to certain drugs, including the 4-aminoquinolines, or to suboptimal drug treatment as occurs during the emergence of drug resistance with other "Parasitemia asexual reproduction" classes, can promote sexual commitment [ 1516 ]. Without tools to study commitment in vivo ie, validated and sensitive sexual ring stage markersdefinitively answering these questions is very difficult.
Piperaquine is a bisquinoline antimalarial that was developed in the s. It was first used as a monotherapy in China, leading to the development of resistance in that country [ 17 ]. However, it shows little cross-resistance with chloroquine, indicating that it is not a substrate for extrusion from the digestive vacuole through the P.
Piperaquine is coformulated with dihydroartemisinin in a widely used artemisinin combination therapy. The experimentally induced blood-stage malaria IBSM infection model [ 23 ] has proven very useful for monitoring the outcomes of different treatments and for informing deployment of those drugs. For example, this model was used to show that monotherapy of P.
However, it was not clear from this study whether these gametocytes were induced by drug treatment or were formed prior to treatment. Plasmodium falciparum gametocyte exported protein-5 PfGEXP5 has been recently identified as a specific marker of early stage sexually differentiated parasites [ 25 ].
In this article, we report that measuring gexp5 transcript levels provides a convenient and reliable method for enumerating ring-stage gametocytes both in vitro and in vivo. Using quantitative reverse transcriptase assays specific for gexp5 gametocyte ringsskeleton-binding protein-1 sbp1 ; Parasitemia asexual reproduction and gametocyte ringsand P.
Samples were harvested during the first cycle after reinvasion, N-acetyl-glucosamine was added to remove asexual stage parasites [ 28 ], and further samples were collected as gametocytogenesis progressed. Blood samples were collected from subjects in the third cohort of a previously reported study whose objective was to investigate the antimalarial activity of piperaquine [ 24 ].
Parasitemia in the clinical trial was monitored using a real-time, quantitative PCR using primers and a hydrolysis probe that targets the 18S rDNA gene [ 29 ]. For quantification of specific parasite gene transcripts, primer pairs and hydrolysis probes were designed to amplify the following transcripts: The assays were designed to exclude amplification of any contaminating genomic DNA targets.
These standard curves were used to report the absolute number of mRNA transcripts in samples by interpolation using a linear regression model of the measured concentration transcripts per microliter of the standard curve material Supplementary Material. Two hours "Parasitemia asexual reproduction" reinvasion, any remaining schizonts were removed, and samples of the culture were harvested at 6, 12, and 24 hpi Figure 1A Parasitemia asexual reproduction, top row.
ASchematic representation of the in vitro commitment experiment. Parasites were harvested at schizont Sch stage cycle 1, 42 hpiand at 6, 12, and 24 hpi. All values shown are normalized to 18S rRNA and expressed as fold-change over the schizont control sample. The mean values and 5—95 percentile error bars are shown. The transcript level normalized to the 18S rRNA signal increases significantly by 6 hours after reinvasion at high density.
The transcript level peaks at 12 hpi, with a fold increase compared with schizont-stage parasites, and remains "Parasitemia asexual reproduction" at 24 hpi.
We subjected the hpi culture to treatment with N-acetyl-glucosamine to remove asexual parasites and examined transcript levels in cultures containing sexual parasites from stage II to stage V of development Figure 1E—G. Levels of the Parasitemia asexual reproduction transcript appear to increase steadily from stage II to stage V of development Figure 1E ; however, it should Parasitemia asexual reproduction pointed out that transcript levels are normalized to the 18S rRNA signal.
Thus, they may appear artificially low in the early stages of gametocyte development due to the presence of nonviable but still present asexual parasites. Nevertheless, the level of gexp5 transcripts was sufficient in parasites beyond Stage I to preclude measurement of this transcript in isolation as a marker the exclusive presence of early stage gametocytes.
In contrast, the sbp1 transcript remains very low across stage II to stage V of sexual development Figure 1F.
These data demonstrate the usefulness Parasitemia asexual reproduction the gexp5 Parasitemia asexual reproduction as a marker for the presence of early-stage gametocytes in mixed populations of asexual and sexual parasites, as well as across the entire developmental phase. By contrast, sbp1which is expressed in both asexual and sexual-stage rings [ 30 ], is not useful in differentiating asexual parasites from early-stage gametocytes in mixed populations. These data indicate that measurement of all 3 of these markers simultaneously will provide a means of determining which lifecycle stages are present in the circulation.
We used these markers to analyze samples collected from a subset of subjects who had participated in a previously reported IBSM study [ 24 ].
Six malaria-naive, healthy male and nonpregnant female subjects aged 18—50 years were infected with the reference strain of P. A typical growth pattern of asexual parasitemia was observed in all study subjects Figure 2Awith parasitemia becoming detectable by qPCR 18S rDNA on day 4 after infection 4 days before treatment. The number of parasite genomes increased Parasitemia asexual reproduction the log sinusoidal pattern that is characteristic of a synchronous infection, with peak periods of circulating rings stages followed by periods of sequestration of mature stage-infected RBCs, then a further increase following schizont rupture.
Three subjects showed recrudescent parasitemia on days 11, 23, and 27 after infection, when they received a second larger dose of piperaquine mg; arrows. A18S genome copies numbers of parasites in the peripheral blood of subjects before and after piperaquine treatment. Levels were interpolated by linear regression analysis using cloned plasmid DNA calibration curves. Dotted lines indicate time of administration of a single mg dose of piperaquine.
Arrows indicate timing of a second dose of piperaquine mg for all except for 1 patient, who received the second dose Parasitemia asexual reproduction day earlier. Standard error of the mean is shown. Negative no detection PCR results are given a value of 1.
Data were reanalyzed from a previous study [ 24 ]. We measured levels of gexp5sbp1, and pfs25 mRNA transcripts in the peripheral blood of the subjects to obtain a more detailed view of the temporal course of transcription of these mRNA markers of asexual and sexual parasites.
By day 4 after infection 4 days before treatmentsbp1 transcript levels were readily detectable, consistent with the Parasitemia asexual reproduction of circulating ring-stage parasites Figure 3A.
Levels of sbp1 transcript increased on day 5, then exhibited peaks and troughs, synchronous with those observed for18S rRNA and rDNA, as the infection progressed.
Again, this is consistent with cycles of asexual replication. The thin dotted lines indicate time of administration of a single mg dose of piperaquine. Arrows indicate timing of a second dose of piperaquine mg for all except for 1 patient, who received their second dose 1 day earlier. A, gexp5 and sbp1. B, pfs25 and sbp1. The production of gametocyte rings appears to occur very early in the blood-stage infection, when the parasite density is very low approximately per milliliter and well before antimalarial treatment with piperaquine was administered.
A trough in gexp5 transcript levels on days 5—6 is consistent with disappearance of the first round of ring-stage gametocytes due to sequestration.