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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Here we report on the effects of sera from atovaquone/ proguanil-treated volunteers on the mosquito transmission of P. falciparum, and on our observation that samples taken up to 6 weeks after treatment inhibited parasite transmission. Further, sera also inhibited asexual blood stage development, again for up to 6 weeks after treatment. It also is established that these effects were replicated by serum samples taken from volunteers who had been given only atovaquone. In contrast to inhibition observed with atovaquone-sensitive strains of P. falciparum, asexual stage development of a recently isolated atovaquone-resistant strain was not affected by the post-treatment sera. These results suggest that anti-parasitic activity persisted in the sera of these volunteers well beyond the clearance time of effective drug concentrations from the plasma expected from published pharmacodynamic data (based on high performance liquid chromatography [HPLC] assays). This information may help in devising more-appropriate formulation and use of MalaroneTM.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Transmission experiments: P falciparum culture. The 3D7 clone of the NF54 strain of P. falciparum (provided by Prof. D. Walliker) was used in all transmission experiments. This was maintained in a semiautomated culture system developed by Ponnudurai et al.8 Briefly, parasites from asexual cultures grown in standard tissue culture flasks (Nunc, Roskilde, Denmark) in RPMI-1640 medium containing 25 mM Hepes, 2 g/L sodium bicarbonate (Sigma-Aldrich, Poole, UK) supplemented with 10% v/v human heat-inactivated AB serum, 50 mg/L hypoxanthine (Sigma-Aldrich), and gassed with 1% oxygen, 3% CO2, and 96% nitrogen were diluted to 1% parasitemias with fresh red cells. Then, 750 µL of these infected red cells were added to each specially designed glass culture flask8 held on a cam-operated table that allowed medium (10 mL) to be changed automatically twice daily without removal from the incubator. Small samples were removed for testing for exflagellation7,9,10 from day 10 onward. When enough mature gametocytes capable of exflagellation were present in cultures, the flasks were harvested, usually on days 11–17; flask contents were centrifuged in tubes prewarmed to 37°C containing 200 µL of fresh red cells previously washed once with phosphate-buffered saline (PBS). After centrifugation for 3 mins at 400g, supernatant culture medium was removed and the cell pellet mixed with respective sera to give a 40% hematocrit, keeping cells and sera at 37°C. Then 500-µL samples of the serum/parasitized red cells were placed in membrane feeders7,9 also kept at 37°C by the passage of warm water.
Mosquito infections. Five- to seven-day-old female Anopheles stephensi mosquitoes were given water only for 24h before infection. Pots containing approximately 50 mosquitoes were placed beneath the membrane feeders, and mosquitoes were allowed to feed for 30 mins (at 21–22°C ambient temperature). The mosquitoes were then held at 27°C, 80% relative humidity overnight, briefly anesthetized with CO2 while unfed mosquitoes were removed, and subsequently maintained in the same conditions on fructose/para-aminobenzoic acid.7,9 On day 7, they were killed; then they were dissected and oocyst counts made on the midguts.
Infectivity was defined as the arithmetic mean oocyst number per mosquito in batches of mosquitoes fed post-treatment sera expressed as a percentage of the equivalent number in those fed pretreatment (day 0) sera;7,10 data on sera of three atovaquone/proguanil-treated volunteers tested in four experiments were pooled and the means for each time point were expressed as the percentage of the mean for the day 0 sera as previously described.7,10 Data from three experiments on one atovaquone-treated volunteer (A1) were pooled but there was only one experiment on A2 sera, with data only up to day 28.
The number of infected mosquitoes in each pot also was noted and was expressed as a percentage of the total number dissected; results for different experiments were again pooled and expressed as a percentage of the day 0 value.
Asexual stage experiments. Three lines of P. falciparum were maintained as asexual stage cultures in standard tissue culture flasks: the 3D7 clone of NF54; a chloroquine-resistant line, K1 (provided by Prof. D. Warhurst); and an atovaquone-resistant isolate (NGATV01) recently isolated from a patient at St. Mary’s Hospital, London.11 The medium and gas mixture were as described above but medium was renewed only once daily, and cultures were diluted with fresh red cells two to three times a week.
For the tests on sera from volunteers, parasitized red cells were diluted to a 1% parasitemia with fresh red cells (previously washed twice with PBS) and incubated for 48 h at a 2–4% hematocrit with 10% v/v day 0 or post-treatment sera in 100-µL cultures in 96-well trays. The trays were held in gas-tight chambers in the usual gas mixture. Parasitemias were counted blind on Giemsa-stained thin blood films, and the number of morphologically normal and abnormal rings, tro-phozoites, and schizonts was noted.
Sera were tested in triplicate and mean parasitemias expressed as a percentage of those on day 0; data from two to three experiments per strain were pooled and the final mean level of parasite growth expressed as a percentage of that at day 0.
Statistical treatment of results. Data (mean oocyst counts, mosquito infections, or percentage parasitemias) from post-treatment sera were compared with data from day 0 sera using the Mann-Whitney U test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sera taken 4–28 days after atovaquone/proguanil treatment totally blocked infection of the mosquitoes, and significant inhibition of oocyst development (P = 0.05; Mann-Whitney U test) was still present on day 42. With day 42 sera, some oocysts were found (mean: 12.5% SEM ± 1.94, of day 0 value), and for day 56 sera, mean oocyst numbers were approaching those of day 0 (87.0% SEM ± 3.70 of day 0; Table 1
). The percentage of infected mosquitoes (prevalence) was much reduced in pots containing post-treatment atovaquone/ proguanil sera up to day 42 (Table 1). Of a total of 315 mosquitoes dissected from all pots fed atovaquone/proguanil sera from day 4 to day 28, no oocyst-positive mosquito was found.
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). No infected mosquitoes were found in pots fed post-treatment atovaquone sera up to day 49 for volunteer A1, although a total of 309 were dissected. Sera from volunteer A2 inhibited infectivity up to day 28. (Ninety-five mosquitoes from post-treatment sera dissected were all negative, compared with 39 of 43 positive for day 0 serum.)
Asexual stage experiments.
After initiation of cultures at 1% and 48-hour culture at 37°C, parasitemias in wells containing day 0 sera ranged from 3–11% (mean 7.6% SEM ± 0.79) for all culture experiments. For the 3D7 clone, post-treatment atovaquone/proguanil sera were inhibitory up to day 28 (mean 11.4% SEM ± 0.3) of growth in day 0 sera, see Figure 1; P = 0.05 by Mann-Whitney U test in all experiments comparing parasitemias in wells containing sera up to and including day 28 with day 0 sera). The level of inhibition was less than that seen with the transmission experiments. This may reflect the 10% v/v of sera in the culture wells compared with the 60% v/v in membrane feeders, but it also should be noted that there is a natural decline in parasite numbers in the mosquito of 40- to 316-fold between macrogamete and ooki-nete.12 However, on day 42, the mean parasitemia still was only 43.9% SEM ± 17.7 of the day 0 value. Of parasites present in wells containing day 7 to day 28 sera, 80–90% were abnormal (pycnotic, irregular cytoplasm, etc.). By day 42, this percentage had fallen to 16–22%. (Data not shown.) Tested against the chloroquine-resistant K1 strain of P. falciparum, a similar pattern of inhibition was observed, i.e., mean growth in wells containing day 7 to day 28 sera was less than 20% of the day 0 value, and parasites in these wells also exhibited abnormal morphology. (Data not shown.) Post-treatment sera from volunteer A1 were inhibitory up to and including day 49 when tested against asexual parasites of the 3D7 clone of P. falciparum (Figure 2): for day 49 sera, growth was a mean of 15.3% SEM ± 3.07 of day 0 sera, Figure 2. In all experiments comparing parasitemias in wells containing sera up to and including day 49 with day 0 sera, the differences were statistically significant (P = 0.05, Mann-Whitney U test). Sera from A2 were less inhibitory by day 28 (40% ± of growth in day 0 sera, Figure 3), though the difference was still significant (P = 0.05, Mann-Whitney U test).
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), except that a late serum (day 42) from A2 reduced growth by 51%. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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With the atovaquone-resistant strain NGATV01, sera from neither donor caused any significant inhibition of growth in vitro, except with the day 42 A2 serum; at this time point, inhibition by atovaquone is unlikely as both sensitive and resistant parasites were affected and other factors in sera can affect parasite development in vitro.12
We previously reported HPLC analysis of atovaquone in serum from atovaquone (A2) sera;7 this fell from 23 µmol/L on day 3 to 7.2 µmol/L on day 7, but on days 14 and 28, it was 1.2 µmol/L, close to the lower limit of quantification of 1.0 µmol/L.13 These results were obtained using an improved HPLC method, but it still may have been insufficiently sensitive to measure very low levels of atovaquone accurately.14 Interestingly, it is known that atovaquone binds to protein, and activity against P. berghei in sera passed through molecular sieves was retained in the high molecular weight fraction—> 10 kDa.7 Although HPLC has replaced biologic assays in many situations, the latter still are used (e.g., for botulinum toxin14). In contrast with the atovaquone results, the kinetics of parasite killing in the mosquito transmission assay for pyrimethamine/sulfadoxine and proguanil reflected exactly the kinetics of drug elimination assessed by HPLC.7,10
A further indication of the sensitivity of the infectivity assay was the observation that a 1:10,000 dilution of day 3 serum did not reduce the inhibition of P. berghei transmission at all,7 and even at a 1:100 dilution, day 21 serum still inhibited transmission by 57%.7 These data demonstrate that the biologic assays for atovaquone, though not quantitative, are considerably more sensitive than physicochemical methods for detecting its presence in serum.
It has been shown that when atovaquone was used alone to treat malaria, resistant parasites rapidly appeared,6,15 hence its formulation in combination with proguanil in MalaroneTM. However, our results show that atovaquone (or theoretically a metabolite, though there is no evidence that it is metabolized in humans; Chulay, personal communication) persists at low concentrations in plasma for a prolonged period after treatment when proguanil is no longer present, thus providing an opportunity for atovaquone-resistant P. falciparum to develop in patients who could be reinfected over this period; the useful life of atovaquone/proguanil in the field may therefore be shortened.
Received April 17, 2002. Accepted for publication August 22, 2002.
Acknowledgments: We thank GlaxoWellcome (now GlaxoSmith-Kline) for the supply of Malarone and atovaquone. We also thank our volunteers who took the drugs and donated sera, and J. Mendoza for keeping us supplied with mosquitoes.
Financial support: Dr. Butcher was supported by GlaxoWellcome (now GlaxoSmithKline).
Reprint requests: Dr. G. Butcher, Department of Biologic Sciences, Imperial College of Science Technology and Medicine, Sir Alexander Fleming Building, Imperial College Road, London SW7 2AZ UK, Telephone: 44-0207-5945381, Fax: 44-0207-5495425, E-mail: g.butcher{at}ic.ac.uk
Authors’ addresses: Dr. G. Butcher, Prof. RE Sinden, Department of Biologic Sciences, Imperial College of Science Technology and Medicine, Sir Alexander Fleming Building, Imperial College Road, London.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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