Effect of Antimalarial Drugs on Plasmodium falciparum Gametocytes

Recent renewed emphasis on the eradication of malaria has highlighted the need for more tools to achieve this ambitious goal. Despite major progress in places where insecticide-impregnated bed nets and artemesinin-based combination therapy have been deployed, an estimated 3.3 billion people remain at risk of malaria [1]. The deployment of an effective vaccine in the near future remains unlikely, which means that vector control and case management by chemotherapy will remain the primary means of control. Once these tools reduce the prevalence of infection to low levels, additional efforts will be required to interrupt transmission

The switch from the asexual intraerythrocytic stage to the sexual stage is essential for transmission of the malaria parasite from human host to mosquito vector. Despite the fact that the first malaria parasite to be observed was an exflagellating male gametocyte, this haploid sexual stage has been significantly less well studied than the replicating asexual stage. Impediments to the study of gametocyte biology have included the inability to readily distinguish early-stage gametocytes from asexually replicating parasites, as well as a lack of methods to physically separate gametocytes from asexual parasites

Of significance to the goal of interrupting transmission is the fact that considerable increases in gametocyte prevalence have been observed after widespread use of some antimalarial therapies (reviewed by Drakeley et al [2]). However, it is not known whether this is the result of increased commitment to gametocytogenesis among the parasites or of posttreatment maturation and survival of existing gametocytes, because gametocyte production is influenced by a range of factors, including the host response [3, 4]. Although the question of drug-induced gametocytogenesis has been investigated in a number of clinical studies (reviewed by Babiker et al. [5]), drawing firm conclusions from these studies has been difficult. It is clear, however, that gametocytemia is a very sensitive indicator of emerging drug resistance [6], with increasing gametocyte prevalence having been shown to precede measurable changes in parasite clearance or a decrease in cure rates [7]. Monitoring the effects of antimalarial chemotherapy on gametocyte carriage and identifying drugs that promote gametocytocidal activity are therefore priorities in efforts to eliminate malaria [7]

However, quantification of gametocytogenesis has been technically challenging because of the difficulty in recognizing early-stage gametocytes and measuring low-level gametocytemia, which is notoriously difficult. In vitro studies aimed at overcoming these technical difficulties have been labor intensive and are not readily adapted for large-scale studies

We have elsewhere reported [8] the development of an assay that uses a green fluorescent protein chimera of the early sexual blood stage protein Pfs16 as a marker for commitment to gametocytogenesis. Analysis of parasites via fluorescence-activated cell sorting (FACS) enables the accurate quantitation of gametocyte production well before the gametocytes are morphologically distinguishable from asexual parasites [8]. In addition, this method enables the processing of large numbers of samples in a relatively short time, which is a potential advantage for high-throughput screening

The aim of the current study in using this novel assay system was to evaluate the effects of commonly used antimalarial drugs on gametocyte production in vitro and to measure the direct activity of these drugs against late-stage gametocytes. This may lead to a better understanding of the effects of antimalarial drugs on the transmission dynamics associated with current treatment regimens

Methods Transgenic chimeric parasites with green fluorescent protein-tagged Pfs16 were generated and maintained as described elsewhere [8]. The susceptibility of the transgenic parasites and of the parental wild-type parasite line 3D7 to chloroquine, quinine, atovaquone, artemisinin, mefloquine, primaquine, piperaquine, and pyronaridine was determined using tritiated hypoxanthine incorporation. The assays were performed in triplicate on at least 2 separate occasions (Figure 1A )

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Figure 1

A, Inhibitory concentrations of the 8 antimalarial agents against 3D7 transgenic Plasmodium falciparum clones expressing green fluorescent protein-tagged Pfs16. These inhibitory concentrations were not statistically significantly different from those obtained for the wild-type parental clone 3D7 (data not shown). B, Change in the number of gametocytes in all of the test and control wells 96 h after the addition of drug pressure. The total change in the gametocyte number was obtained by subtracting the initial counts from the final counts. The 8 antimalarial agents were used at the 10%, 50%, and 90% inhibitory concentrations (IC₁₀, IC₅₀, and IC₉₀, respectively) as determined against hypoxanthine incorporation-tested asexually replicating parasites. The bar labeled “C” represents the control wells. Asterisks indicate statistically significant changes in gametocyte numbers when compared with control wells (P<.01)

A single culture of ring-stage transgenic parasites at 0.3% parasitemia and 5% hematocrit was then dispensed in 200-μL aliquots into either control or test wells. The control wells were maintained without drug pressure, and the individual test wells were exposed to the 10%, 50%, and 90% inhibitory concentrations (IC₁₀, IC₅₀, and IC₉₀, respectively) of chloroquine, quinine, atovaquone, artemisinin, mefloquine, primaquine, piperaquine, and pyronaridine in 96-well microtiter plates (final volume, 250 μL) for 48 h. FACS analysis was performed at time 0 to quantitate the initial gametocyte count and then again at 96 h to provide the final gametocyte count. Analysis was performed using a FACScaliber flow cytometer (Becton Dickson) and CellQuest Pro software (BD Biosciences). For this analysis, 500,000 discrete events were counted (including noninfected erythrocytes and erythrocytes infected with either asexual parasites or gametocytes). To obtain the change in gametocyte counts during the assay, the initial gametocyte counts were subtracted from the final gametocyte counts to give the overall increase in the number of gametocytes. Assays were performed in triplicate on 3 separate occasions. Statistical analysis was performed using 1-way analysis of variance (ANOVA)

The direct effect of antimalarial drugs on late-stage (stage IV and V) gametocytes was also determined. Gametocytes were cultured and purified as described elsewhere [9] and maintained in culture for a further 5 d before being tested in the cytotoxicity assay. Identical aliquots of gametocyte culture were then incubated with phosphate-buffered saline (control wells) or antimalarial drugs at concentrations corresponding to the IC₁₀, IC₅₀, and IC₉₀ values that were obtained against asexual parasites for 24 h and then analyzed as described elsewhere [8]. Equivalent amounts of culture from each of the test and control wells were analyzed. Hydroethidine was incorporated into the cultures for the last 24 h because it is converted by metabolizing cells to ethidium, a nucleic acid fluorochrome [10] that allows differentiation between actively metabolizing and nonviable cells. The viability of the late-stage gametocytes was determined for each drug and drug concentration and then compared with the viability of the late-stage gametocytes in the control cultures. The results of all assays were combined and analyzed by means of 1-way ANOVA

Results A total of 8 antimalarial drugs were tested for their effect on gametocytogenesis. Each drug was assessed at 3 different concentrations, and although the change in gametocyte numbers was relatively small, 8 of the 24 treatment regimens caused a statistically significant change in gametocytogenesis, compared with the control cultures (Figure 1B ). Interestingly, each of the 8 drugs, when used at IC₁₀, resulted in increased gametocyte formation, although only 4 drugs (mefloquine, chloroquine, primaquine, and pyronaridine) statistically significantly increased the number of gametocytes (P<.01). At IC₅₀, quinine, artemisinin, and piperaquine each caused a statistically significant increase in the number of gametocytes (P<.01), whereas at IC₉₀, only atovaquone showed any statistically significant effect on gametocyte formation

Although all of the antimalarial drugs that were tested did produce statistically significant increases in gametocyte formation in at least 1 of the drug concentrations used, only a slight dose-dependent effect on the viability of late-stage gametocytes was observed in at least 1 of the 3 concentrations used. When these drugs were tested at concentrations that result in 10%, 50%, or 90% asexual parasite growth inhibition, there was some apparent reduction in gametocyte numbers that appeared to be dose dependent in comparison with the control lines. However, this reduction was not statistically significant (Figure 2)

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Figure 2

Viability of stage IV and V gametocytes for each of the 8 antimalarial drugs after 24 h of drug pressure. Each drug was administered at 10%, 50%, and 90% inhibitory concentrations (IC₁₀, IC₅₀, and IC₉₀, respectively) as determined by means of tritiated hypoxanthine incorporation against asexually replicating parasites. Total numbers were obtained by analyzing equal volumes of each of the test and control wells by means of fluorescence-activated cell sorting as determined by hydroethidine conversion to ethidium in individual parasites

Discussion Initiatives such as the Roll Back Malaria campaign are now starting to have a significant effect on malaria-related morbidity and mortality [1]. Although previous studies have shown that some antimalarial drugs appear to increase gametocytogenesis [11, 12], to our knowledge the present study is the first to undertake a systematic investigation of the effect of antimalarial drugs on gametocytogenesis and gametocyte survival. Unfortunately, the antifolate drugs, such as sulphadoxine and proguanil, could not be tested, because the transgenic parasite clone that was used has altered sensitivity to folate antagonists

All of the 8 drugs evaluated in this study led to a statistically significant increase in gametocyte numbers, at drug doses resulting in inhibition of asexual parasite growth, in at least 1 of the 3 inhibitory concentrations tested. This increase appeared to be drug dependent rather than dose dependent. Nonetheless, all drugs appeared to lead to a small increase in the number of gametocytes when used at the lowest concentrations (IC₁₀), although this increase was significant for only 4 (mefloquine, chloroquine, primaquine, and pyronaridine) of the 8 drugs tested. This result may indicate that these antimalarial drugs, in particular the long-acting quinoline antimalarial drugs, are having an effect on gametocyte formation that is separate from their toxic effect on the parasite. An increase in the spread of drug-resistant parasites within the population may be a consequence of induction of gametocytogenesis that is independent of any significant gametocytocidal effect on blood-stage parasites. This effect warrants further investigation. Although the assay was undertaken in a parasite strain that was drug susceptible, it would be expected that similar results would be observed in drug-resistant parasites, raising the question that therapy may increase transmission if the parasites are drug resistant. Studies investigating the antimalarial activity of drug combinations may also be warranted

Although there appeared to be a trend toward gametocyte induction associated with all of the drugs tested when they were used at IC₁₀ (which may indicate a stress response), at IC₅₀ only cultures treated with quinine, artemisinin, or piperaquine showed a statistically significant increase in gametocytogenesis. This response was not seen when these drugs were used at IC₉₀, which indicates that effective treatment is unlikely to increase gametocyte carriage. However, these findings do support the idea that drug resistance may lead to increases in gametocyte carriage and hence may enhance the spread of resistant parasites

Exposure to only 1 drug, atovaquone, resulted in a statistically significant increase in gametocyte numbers at IC₉₀. However, atovaquone is never used alone but always in combination with proguanil. Although it is possible that this combination therapy may mask the effects of the increase in gametocyte numbers in patients undergoing drug treatment, it was not possible to test this hypothesis because proguanil is an inhibitor of folate metabolism in the parasite

The antimalarial sensitivity of late-stage gametocytes was also investigated. This is the stage of the parasite that remains in the host's circulation for some time while it awaits ingestion during the mosquito blood meal. It has been reported that late-stage gametocytes (stage III onward) become insensitive to some common antimalarial agents [13]. Our results indicate that late-stage gametocytes are refractory to all of the classes of antimalarial agents that were tested in this study. However, we observed a weak trend in dose-dependent reduction of gametocyte numbers in the treatment groups, even though there was no statistically significant difference between the viability of treated gametocytes and that of untreated gametocytes. Because the drug concentrations observed in patients who undergo antimalarial therapy are likely to be significantly higher than the concentrations tested in these studies, it is also likely that some gametocytocidal activity does occur in vivo. In particular, primaquine has been reported to kill mature-stage gametocytes [6]. Although we did not observe any statistically significant effect on late-stage gametocytes at the concentrations that we tested, this observation may be explained by the hypothesis that 1 or more metabolites of primaquine are responsible for its observed gametocidal activity in vivo [14]. It is also possible that drug-treated gametocytes may appear to be metabolically active, as defined by production of the fluorochrome, but may no longer be infectious to mosquitoes because of the effects of the drugs. This possibility warrants further investigation

Both in vivo and in vitro methods are traditionally used to assess the susceptibility of malaria parasites to antimalarial drugs. An advantage of in vitro testing is the ability to measure the susceptibility of Plasmodium falciparum parasites to antimalarial drugs in the absence of confounding host factors, such as immunity. However, in vitro methods have limitations, particularly when it is a metabolite that is believed to be responsible for the antimalarial activity, as is likely to be the case for primaquine [14]. It may be possible to adapt this assay for ex vivo studies using patient serum samples to investigate the effects of drug metabolites on gametocyte production and viability

Drugs against malaria commonly target the asexual parasite, thus relieving the clinical manifestations of the disease. Relatively little data are available on the effects of these drugs on gametocyte formation, as well as their ability to block transmission by killing the late-stage gametocyte. If malaria is to be eliminated, drugs that not only kill the asexual parasite but also block transmission by either reducing gametocyte formation or killing the late-stage gametocyte need to be identified

• Received March 10, 2009.
• Accepted June 19, 2009.

• © 2009 by the Infectious Diseases Society of America