• View in gallery
    Figure 1.

    Mean parasitemias of Plasmodium falciparum cultured 48h in the presence of sera from atovaquone/proguanil-treated volunteers expressed as a percentage of mean pretreatment day 0 sera (±SEM); data from three experiments pooled.

  • View in gallery
    Figure 2.

    Mean parasitemias of Plasmodium falciparum cultured 48h in the presence of sera from atovaquone-treated volunteer A1 tested against the sensitive 3D7 and resistant NGATV01 strains expressed as a percentage of mean pretreatment day 0 sera (±SEM); pooled data from two experiments for each parasite strain.

  • View in gallery
    Figure 3.

    Mean parasitemias of Plasmodium falciparum cultured 48h in the presence of sera from atovaquone-treated volunteer A2 tested against the sensitive 3D7 and resistant NGATV01 strains expressed as a percentage of mean pretreatment day 0 sera (±SEM); pooled data from two experiments for each parasite strain.

  • 1

    Peters W, 1984. Use of drug combinations. Peters W, Richards WHG, eds. Antimalarial Drugs, vol 2. London: Springer-Verlag, 499–509.

  • 2

    Butcher G, 1997. Antimalarial drugs and the mosquito transmission of Plasmodium. Int J Parasitol 27 :975–987.

  • 3

    Sabchereon A, Attanath P, Phanuasksook P, Chanthavanich P, Poonpanich T, Mookmanee D, Chongsuphajaisiddhi T, Sadler BM, Hussein Z, Canfield CJ, Hutchinson DBA, 1998. Efficacy and pharmacokinetics of atovaquone and proguanil in children with multi-drug resistant Plasmodium falciparum malaria. Trans R Soc Trop Med Hyg 92: 201–206.

    • Search Google Scholar
    • Export Citation
  • 4

    Shanks GD, Kremsner PG, Such TY, van der Berg J, Shapiro TA, Scott TR, Chulay JD, 1999. Atovaquone and proguanil hydrochloride (MALARONE™) for prophylaxis of malaria. J Travel Med 6 (Suppl 1) :S21–S27.

    • Search Google Scholar
    • Export Citation
  • 5

    Beerahee M, 1999. Clinical pharmacology of atovaquone and proguanil hydrochloride. J Travel Med 6 (Suppl 1) :S13–S17.

  • 6

    Hudson AT, 1993. Atovaquone—a novel broad-spectrum anti-infective drug. Parasitol Today 9 :66–68.

  • 7

    Butcher GA, Mendoza J, Sinden RE, 2000. Inhibition of the mosquito transmission of Plasmodium berghei by Malarone™ (atovaquone/proguanil). Ann Trop Med Parasitol 94: 429–436

    • Search Google Scholar
    • Export Citation
  • 8

    Ponnudurai TV, Lensen AHW, Leeuwenberg ADEM, Meuwissen JHET, 1982. Cultivation of fertile Plasmodium falciparum gametocytes in semi-automated systems. Trans R Soc Trop Med Hyg 76 :242–250.

    • Search Google Scholar
    • Export Citation
  • 9

    Butcher GA, Sinden RE, Billker O, 1996. Plasmodium berghei: infectivity of mice to Anopheles stephensi mosquitoes. Exp Parasitol 84 :371–379.

    • Search Google Scholar
    • Export Citation
  • 10

    Høgh B, Gamage-Mendis A, Butcher GA, Thompson R, Begtrup K, Mendis C, Enosse SM, Dgedge M, Barreto J, Eling W, Sinden RE, 1998. The differing impact of chloroquine and pyrimethamine/sulfadoxine upon the infectivity of malaria spp. to the mosquito vector. Am J Trop Med Hyg 58 :176–182.

    • Search Google Scholar
    • Export Citation
  • 11

    Fivelman QL, Butcher GA, Adagu IS, Warhurst DC, Pasvol G, 2002. Malarone treatment failure and in vitro confirmation of resistance of Plasmodium falciparum isolate from Lagos, Nigeria. Malaria J 1 :1–4.

    • Search Google Scholar
    • Export Citation
  • 12

    Butcher GA, Clark IA, Crane G, 1987. Inhibition of intraerythrocytic growth of P. falciparum by human sera from Papua New Guinea. Trans R Soc Trop Med Hyg 81 :568–572.

    • Search Google Scholar
    • Export Citation
  • 13

    Lindegardh N, Fundling L, Bergvist Y, 2001. Automated solid-phase extraction method for the determination of atovaquone in capillary blood applied onto sampling paper by rapid high-performance liquid chromatography. J Chromatog B: Biomed Sci Appl 758 :137–144.

    • Search Google Scholar
    • Export Citation
  • 14

    Huang W, Foster JA, Rogchefsky AS, 2000. Pharmacology of botulinum toxin. J Am Acad Dermatol 43 :249–259.

  • 15

    Looareesuwan S, Viravin C, Webster HK, Kyle DE, Hutchinson DB Canfield CJ. 1996. Clinical studies of atovaquone, alone or in combination with other antimalarial drugs, for the treatment of acute uncomplicated malaria in Thailand. Am J Trop Med Hyg 54: 52–56.

    • Search Google Scholar
    • Export Citation
Past two years Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 145 71 19
PDF Downloads 46 35 5
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

 

 

PERSISTENCE OF ATOVAQUONE IN HUMAN SERA FOLLOWING TREATMENT: INHIBITION OF P. FALCIPARUM DEVELOPMENT IN VIVO AND IN VITRO

GEOFF A. BUTCHERDepartment of Biologic Sciences, Imperial College of Science Technology and Medicine, London

Search for other papers by GEOFF A. BUTCHER in
Current site
Google Scholar
PubMed
Close
and
ROBERT E. SINDENDepartment of Biologic Sciences, Imperial College of Science Technology and Medicine, London

Search for other papers by ROBERT E. SINDEN in
Current site
Google Scholar
PubMed
Close

Published pharmacokinetic data indicate that after treatment of patients with therapeutic doses of atovaquone/proguanil hydrochloride (Malarone™, GlaxoSmithKline Research Triangle Park, NC), the plasma half-lives of these drugs are 70h and 15h, respectively. However, using two biologic assays (mosquito transmission and in vitro asexual stage development), we demonstrate here that sera from volunteers treated with atovaquone/proguanil retained activity against Plasmodium falciparum up to 6 weeks after such treatment. This activity was due to atovaquone, as administration of this drug alone replicated the data obtained with the combination. Most notably, asexual stage development of an atovaquone-resistant strain (NGATV01) of P. falciparum was not inhibited by sera taken after atovaquone treatment. These data indicate that for atovaquone, biologic assays, though not quantitative, are more sensitive than the usual physicochemical assays. Also, persistence of atovaquone in plasma at low concentrations for long periods may increase the risk of resistant parasites arising.

INTRODUCTION

To reduce the risk of drug resistance, new antimalarials should target more than one aspect of Plasmodium metabolism,1 be excreted rapidly,1 and, ideally, also inhibit mosquito-transmission.2 A combination of proguanil and atovaquone (Malarone™) appears to fulfill these requirements, having been used therapeutically against drug-resistant Plasmodium falciparum3 and as a prophylactic.4 Cycloguanil, generated from proguanil, inhibits folate metabolism via dihydrofolate reductase, and atovaquone acts on mitochondrial metabolism, inhibiting dihydroorotate dehydrogenase.5 Pharmacodynamic data on proguanil and atovaquone indicated that both are rapidly excreted: The reported plasma half-life of proguanil is 15 hours and that of atovaquone is 70 hours.3,5,6

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 Malarone™.

MATERIALS AND METHODS

Drug treatment of volunteers.

Permission from the Ethics Committee of the Imperial College School of Medicine at St Mary’s Hospital, London, was obtained for drug treatment of volunteers, none of whom had previously experienced malaria. Three volunteers were bled for pretreatment serum and on the same day, day 0, were treated with atovaquone/ proguanil hydrochloride (four tablets, each containing 250 mg atovaquone and 100 mg proguanil daily as a single dose for 3 days); they were subsequently bled for serum 7, 14, 21, 28, 42, and 56 days after day 0. These sera had previously been tested against P. berghei.7 Sera also were obtained from two volunteers (A1 and A2) treated with atovaquone only (Wellvone™ [GlaxoWellcome Research Triangle Park, NC] at a dosage equivalent to that in atovaquone/proguanil) taken on day 0 and on days 7, 14, 21, 28 or 35, and 42 or 49 after treatment.

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.

RESULTS

Transmission experiments.

The numbers of fed mosquitoes per pot dissected 7 days after feeding usually ranged from about 20–40. In pots containing mosquitoes fed control day 0 sera from the atovaquone/proguanil-treated volunteers, the mean oocyst count per mosquito was 13.2 (range 0.3–33.2) and for the atovaquone serum controls, it was 7.1 (range 0.2–13.2). The mean percentage of infected mosquitoes for day 0 sera was 60.3 for atovaquone/proguanil treatment (range 13–83) and for atovaquone treatment was 53.8 (range 14–91).

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.

The prolonged inhibition of infectivity was due to atovaquone: sera taken from both volunteers treated with atovaquone alone totally inhibited oocyst development up to and including day 28 (Table 1). 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).

Tested against resistant clone NGATV01, despite minor inhibition by some sera, there was no significant inhibition of growth by post-treatment sera from either of the atovaquone-treated donors (Figures 2 and 3), except that a late serum (day 42) from A2 reduced growth by 51%.

DISCUSSION

Inhibition of both oocyst development and asexual blood stage growth in vitro of P. falciparum by sera from volunteers treated with atovaquone/proguanil persisted for up to 6 weeks after treatment against the sensitive clone 3D7 as well as a sensitive chloroquine-resistant strain. With volunteer A1, who received atovaquone only, inhibitory activity persisted in sera taken up to 7 weeks after treatment, though volunteer A2 appeared to clear the atovaquone somewhat more rapidly, with reduced inhibition of asexual blood stage growth by day 28 serum and none with day 42 serum when comparing resistant and sensitive parasites. These results are entirely consistent with previously reported data on the effects of these sera on P. berghei.7 Previous experiments on the rodent parasite also demonstrated that sera from a proguanil-treated volunteer became non-inhibitory within a week of treatment.7

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 Malarone™. 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.

Table 1

Mean oocyst numbers in Anopheles stephensi mosquitoes and mean percentage of infected mosquitoes fed Plasmodium falciparum gametocytes in the presence of sera from atovaquone/proguanil and atovaquone-treated volunteers expressed as a percentage of mean pretreatment day 0 sera (±SEM)

Day of sample Atovaquone/proguanil mean % infectivity1 Atovaquone/proguanil mean % mosquito prevalence2 Atovaquone mean % infectivity3 Atovaquone mean % mosquito prevalence4
1 Mean Percentage Infectivity: arithmetic mean oocyst number/mosquito expressed as a percentage of that at day 0; data pooled from four experiments on atovaquone/proguanil sera.
2 Mean Percentage Prevalence: arithmetic mean prevalence of infected mosquitoes expressed as a percentage of that at day 0; data pooled from four experiments on atovaquone/ proguanil sera.
3 Mean Percentage Infectivity: arithmetic mean oocyst number/mosquito expressed as a percentage of that at day 0; atovaquone sera, data pooled from three experiments for A1 and one for A2.
4 Mean Percentage Prevalance: arithmetic mean prevalence of infected mosquitoes expressed as a percentage of that at day 0; atovaquone sera, data pooled from three experiments for A1 and one for A2.
5Data for days 35 and 49 for A1 sera only.
0 100 100 100 100
4 0 0 ND ND
7 0 0 0 0
14 0 0 0 0
21 0 0 0 0
28 0 0 0 0
35 ND ND 05 05
42 12.5 (±1.94) 51.3 (±16.31) ND ND
49 ND ND 05 05
56 87.0 (±3.70) 119.0 (±38.89) ND ND
Figure 1.
Figure 1.

Mean parasitemias of Plasmodium falciparum cultured 48h in the presence of sera from atovaquone/proguanil-treated volunteers expressed as a percentage of mean pretreatment day 0 sera (±SEM); data from three experiments pooled.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 68, 1; 10.4269/ajtmh.2003.68.111

Figure 2.
Figure 2.

Mean parasitemias of Plasmodium falciparum cultured 48h in the presence of sera from atovaquone-treated volunteer A1 tested against the sensitive 3D7 and resistant NGATV01 strains expressed as a percentage of mean pretreatment day 0 sera (±SEM); pooled data from two experiments for each parasite strain.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 68, 1; 10.4269/ajtmh.2003.68.111

Figure 3.
Figure 3.

Mean parasitemias of Plasmodium falciparum cultured 48h in the presence of sera from atovaquone-treated volunteer A2 tested against the sensitive 3D7 and resistant NGATV01 strains expressed as a percentage of mean pretreatment day 0 sera (±SEM); pooled data from two experiments for each parasite strain.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 68, 1; 10.4269/ajtmh.2003.68.111

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).

REFERENCES

  • 1

    Peters W, 1984. Use of drug combinations. Peters W, Richards WHG, eds. Antimalarial Drugs, vol 2. London: Springer-Verlag, 499–509.

  • 2

    Butcher G, 1997. Antimalarial drugs and the mosquito transmission of Plasmodium. Int J Parasitol 27 :975–987.

  • 3

    Sabchereon A, Attanath P, Phanuasksook P, Chanthavanich P, Poonpanich T, Mookmanee D, Chongsuphajaisiddhi T, Sadler BM, Hussein Z, Canfield CJ, Hutchinson DBA, 1998. Efficacy and pharmacokinetics of atovaquone and proguanil in children with multi-drug resistant Plasmodium falciparum malaria. Trans R Soc Trop Med Hyg 92: 201–206.

    • Search Google Scholar
    • Export Citation
  • 4

    Shanks GD, Kremsner PG, Such TY, van der Berg J, Shapiro TA, Scott TR, Chulay JD, 1999. Atovaquone and proguanil hydrochloride (MALARONE™) for prophylaxis of malaria. J Travel Med 6 (Suppl 1) :S21–S27.

    • Search Google Scholar
    • Export Citation
  • 5

    Beerahee M, 1999. Clinical pharmacology of atovaquone and proguanil hydrochloride. J Travel Med 6 (Suppl 1) :S13–S17.

  • 6

    Hudson AT, 1993. Atovaquone—a novel broad-spectrum anti-infective drug. Parasitol Today 9 :66–68.

  • 7

    Butcher GA, Mendoza J, Sinden RE, 2000. Inhibition of the mosquito transmission of Plasmodium berghei by Malarone™ (atovaquone/proguanil). Ann Trop Med Parasitol 94: 429–436

    • Search Google Scholar
    • Export Citation
  • 8

    Ponnudurai TV, Lensen AHW, Leeuwenberg ADEM, Meuwissen JHET, 1982. Cultivation of fertile Plasmodium falciparum gametocytes in semi-automated systems. Trans R Soc Trop Med Hyg 76 :242–250.

    • Search Google Scholar
    • Export Citation
  • 9

    Butcher GA, Sinden RE, Billker O, 1996. Plasmodium berghei: infectivity of mice to Anopheles stephensi mosquitoes. Exp Parasitol 84 :371–379.

    • Search Google Scholar
    • Export Citation
  • 10

    Høgh B, Gamage-Mendis A, Butcher GA, Thompson R, Begtrup K, Mendis C, Enosse SM, Dgedge M, Barreto J, Eling W, Sinden RE, 1998. The differing impact of chloroquine and pyrimethamine/sulfadoxine upon the infectivity of malaria spp. to the mosquito vector. Am J Trop Med Hyg 58 :176–182.

    • Search Google Scholar
    • Export Citation
  • 11

    Fivelman QL, Butcher GA, Adagu IS, Warhurst DC, Pasvol G, 2002. Malarone treatment failure and in vitro confirmation of resistance of Plasmodium falciparum isolate from Lagos, Nigeria. Malaria J 1 :1–4.

    • Search Google Scholar
    • Export Citation
  • 12

    Butcher GA, Clark IA, Crane G, 1987. Inhibition of intraerythrocytic growth of P. falciparum by human sera from Papua New Guinea. Trans R Soc Trop Med Hyg 81 :568–572.

    • Search Google Scholar
    • Export Citation
  • 13

    Lindegardh N, Fundling L, Bergvist Y, 2001. Automated solid-phase extraction method for the determination of atovaquone in capillary blood applied onto sampling paper by rapid high-performance liquid chromatography. J Chromatog B: Biomed Sci Appl 758 :137–144.

    • Search Google Scholar
    • Export Citation
  • 14

    Huang W, Foster JA, Rogchefsky AS, 2000. Pharmacology of botulinum toxin. J Am Acad Dermatol 43 :249–259.

  • 15

    Looareesuwan S, Viravin C, Webster HK, Kyle DE, Hutchinson DB Canfield CJ. 1996. Clinical studies of atovaquone, alone or in combination with other antimalarial drugs, for the treatment of acute uncomplicated malaria in Thailand. Am J Trop Med Hyg 54: 52–56.

    • Search Google Scholar
    • Export Citation

Footnotes

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.

Author Notes

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@ic.ac.uk
Save