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Effect of Antimalarial Drugs on Plasmodium falciparum Gametocytes Christopher L. Peatey,1,3 Tina S. Skinner-Adams,1,4 Matthew W. A. Dixon,1,5 James S. McCarthy,2,4 Donald L. Gardiner,1,4,a and Katharine R. Trenholme1,4,a 1 Malaria Biology Laboratory and 2Clinical Tropical Medicine Laboratory, Queensland Institute of Medical Research, Herston, 3School of Chemistry and Molecular Biosciences and 4School of Medicine, University of Queensland, Brisbane, and 5La Trobe University, Bundoora, Victoria, Australia
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 prevReceived 10 March 2009; accepted 19 June 2009; electronically published 21 October 2009. Potential conflicts of interest: none reported. Financial support: National Health and Medical Research Council of Australia (grants to D.L.G., J.M.C., and K.R.T.); ANZ (Trustees PhD scholarship to M.W.A.D.); Australian Department of Education, Employment and Workplace Relations (Australian Postgraduate Award to C.L.P.); University of Queensland (Dr Diana Cavaye Scholarship to C.L.P.); Tudor Foundation (grants to D.L.G. and K.R.T.). a D.L.G. and K.R.T. contributed equally to this work. Reprints or correspondence: Dr Don Gardiner, Malaria Biology Laboratory, Queensland Inst of Medical Research, Herston 4006, Australia (
[email protected]). The Journal of Infectious Diseases 2009; 200:1518–21 2009 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2009/20010-0006$15.00 DOI: 10.1086/644645
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Gametocytes are the sexual stage of the malaria parasite and are essential for transmission to the mosquito. Antimalarial drugs have been reported to affect gametocyte production in vivo, which leads to a potential increase in transmission. We used transgenic Plasmodium falciparum parasites expressing a green fluorescent protein tag in a fluorescence-activated cell sorting–based assay to measure the effect of 8 antimalarial drugs on gametocyte production in vitro. Exposure to antimalarial drugs resulted in an increase in the number of gametocytes in test cultures. Although a dose-dependent reduction in late-stage gametocyte viability was observed, none of the drugs tested statistically significantly reduced gametocyte numbers.
alence 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 druginduced 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
formed 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 cul-
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). BRIEF REPORT • JID 2009:200 (15 November) • 1519
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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). A single culture of ring-stage transgenic parasites at 0.3% parasitemia and 5% hematocrit was then dispensed in 200-mL 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 mL) 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 per-
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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.
sequence 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.
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tures. 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 dosedependent 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). Discussion. Initiatives such as the Roll Back Malaria campaign are now starting to have a significant effect on malariarelated 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 con-
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.
References 1. World Health Organization (WHO). World malaria report 2008. WHO Web site. http://www.who.int/malaria/wmr2008/malaria2008.pdf. Geneva: World Health Organization, 2008. 2. Drakeley C, Sutherland C, Bousema JT, Sauerwein RW, Targett AT. The epidemiology of Plasmodium falciparum gametocytes: weapons of mass dispersion. Trends Parasitol 2006; 22:424–30. 3. Smalley ME, Brown J. Plasmodium falciparum gametocytogenesis stimulated by lymphocytes and serum from infected Gambian children. Trans R Soc Trop Med Hyg 1981; 75:316–7. 4. Ono T, Nakai T, Nakabayashi T. Induction of gametocytogenesis in Plasmodium falciparum by the culture supernatant of hybridoma cells producing anti–P. falciparum antibody. Biken J 1986; 29:77–81. 5. Babiker HA, Schneider P, Reece SE. Gametocytes: insights gained during a decade of molecular monitoring. Trends Parasitol 2008; 24:525–30. 6. White NJ. The role of anti-malarial drugs in eliminating malaria. Malar J 2008; 7(Suppl 1):S8. 7. Greenwood BM. Control to elimination: implications for malaria research. Trends Parasitol 2008; 24:449–54. 8. Dixon MW, Peatey CL, Gardiner DL, Trenholme KR. A green fluorescent protein-based assay for determining gametocyte production in Plasmodium falciparum. Mol Biochem Parasitol 2009; 163:123–6. 9. Muhia DK, Swales CA, Deng W, Kelly JM, Baker DA. The gametocyte activating factor xanthurenic acid stimulates an increase in membraneassociated guanylyl cyclise activity in the human malaria parasite Plasmodium falciparum. Mol Microbiol 2001; 42:553–60. 10. van der Heyde HC, Elloso MM, vande Waa J, Schell K, Weidanz WP. Use of hydroethidine and flow cytometry to assess the effects of leukocytes on the malarial parasite Plasmodium falciparum. Clin Diagn Lab Immunol 1995; 2:417–25. 11. Buckling A, Ranford-Cartwright LC, Miles A, Read AF. Chloroquine increases Plasmodium falciparum gametocytogenesis in vitro. Parasitology 1999; 118:339–46. 12. Puta C, Manyando C. Enhanced gametocyte production in Fansidartreated Plasmodium falciparum malaria patients: implications for malaria transmission control programmes. Trop Med Int Health 1997; 2: 227–9. 13. Lang-Unnasch N, Murphy AD. Metabolic changes of the malaria parasite during the transition from the human to the mosquito host. Annu Rev Microbiol 1998; 52:561–90. 14. Bates MD, Meshnick SR, Sigler CI, Leland P. In vitro effects of primaquine and primaquine metabolites on exoerythrocytic stages of Plasmodium berghei. Am J Trop Med Hyg 1990; 42:532–7.
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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 latestage 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
