Atovaquone-Proguanil in Combination With Artesunate to Treat Multidrug-Resistant P. falciparum Malaria in Cambodia: An Open-Label Randomized Trial

Mariusz Wojnarski, Chanthap Lon, Pattaraporn Vanachayangkul, Panita Gosi, Somethy Sok, Agus Rachmat, Dustin Harrison, Catherine M Berjohn, Michele Spring, Suwanna Chaoratanakawee, Mali Ittiverakul, Nillawan Buathong, Soklyda Chann, Saowaluk Wongarunkochakorn, Andreea Waltmann, Worachet Kuntawunginn, Mark M Fukuda, Hana Burkly, Vireak Heang, Thay Keang Heng, Nareth Kong, Threechada Boonchan, Bolin Chum, Philip Smith, Andrew Vaughn, Satharath Prom, Jessica Lin, Dysoley Lek, David Saunders, Mariusz Wojnarski, Chanthap Lon, Pattaraporn Vanachayangkul, Panita Gosi, Somethy Sok, Agus Rachmat, Dustin Harrison, Catherine M Berjohn, Michele Spring, Suwanna Chaoratanakawee, Mali Ittiverakul, Nillawan Buathong, Soklyda Chann, Saowaluk Wongarunkochakorn, Andreea Waltmann, Worachet Kuntawunginn, Mark M Fukuda, Hana Burkly, Vireak Heang, Thay Keang Heng, Nareth Kong, Threechada Boonchan, Bolin Chum, Philip Smith, Andrew Vaughn, Satharath Prom, Jessica Lin, Dysoley Lek, David Saunders

Abstract

Background: Recent artemisinin-combination therapy failures in Cambodia prompted a search for alternatives. Atovaquone-proguanil (AP), a safe, effective treatment for multidrug-resistant Plasmodium falciparum (P.f.), previously demonstrated additive effects in combination with artesunate (AS).

Methods: Patients with P.f. or mixed-species infection (n = 205) in Anlong Veng (AV; n = 157) and Kratie (KT; n = 48), Cambodia, were randomized open-label 1:1 to a fixed-dose 3-day AP regimen +/-3 days of co-administered artesunate (ASAP). Single low-dose primaquine (PQ, 15 mg) was given on day 1 to prevent gametocyte-mediated transmission.

Results: Polymerase chain reaction-adjusted adequate clinical and parasitological response at 42 days was 90% for AP (95% confidence interval [CI], 82%-95%) and 92% for ASAP (95% CI, 83%-96%; P = .73). The median parasite clearance time was 72 hours for ASAP in AV vs 56 hours in KT (P < .001) and was no different than AP alone. At 1 week postprimaquine, 7% of the ASAP group carried microscopic gametocytes vs 29% for AP alone (P = .0001). Nearly all P.f. isolates had C580Y K13 propeller artemisinin resistance mutations (AV 99%; KT 88%). Only 1 of 14 treatment failures carried the cytochrome bc1 (Pfcytb) atovaquone resistance mutation, which was not present at baseline. P.f. isolates remained atovaquone sensitive in vitro but cycloguanil resistant, with a triple P.f. dihydrofolate reductase mutation.

Conclusions: Atovaquone-proguanil remained marginally effective in Cambodia (≥90%) with minimal Pfcytb mutations observed. Treatment failures in the presence of ex vivo atovaquone sensitivity and adequate plasma levels may be attributable to cycloguanil and/or artemisinin resistance. Artesunate co-administration provided little additional blood-stage efficacy but reduced post-treatment gametocyte carriage in combination with AP beyond single low-dose primaquine.

Keywords: artesunate; atovaquone-proguanil; drug resistance; malaria; primaquine.

Published by Oxford University Press on behalf of Infectious Diseases Society of America 2019.

Figures

Figure 1.
Figure 1.
CONSORT flow diagram. Abbreviations: AP, atovaquone-proguanil; ASAP, artesunate-atovaquone-proguanil; AV, Anlong Veng; FU, follow-up; KT, Kratie; PQ, primaquine.
Figure 2.
Figure 2.
Clinical efficacy. Kaplan-Meier survival analysis of artesunate-atovaquone-proguanil (ASAP) efficacy over 42 days vs atovaquone-proguanil (AP) alone at 2 sites in Cambodia based on number of volunteers remaining malaria free at each interval (inset). A, Overall (log-rank P value for difference = .617). B, Anlong Veng site. C, Kratie site. There were no statistical differences in outcomes for AP (P = .663) or ASAP (P = .317) by location.
Figure 3.
Figure 3.
Parasite clearance times (A) and parasite clearance half-life (B) in Northern (Anlong Veng) and Eastern (Kratie) Cambodia by treatment regimen. All patients with treatment failure assigned to ASAP had a parasite clearance half-life (PCT1/2) >5 hours. Red dots represent parasite recrudescence based on genotyping with msp1, msp2, and glurp polymorphisms. Clear dots represent parasites with the K13 propeller C580Y mutation, green dots represent K13 wild-type parasites, and blue dots represent K13 propeller status unknown. The orange error bars represent medians and interquartile ranges for the variables presented. Comparison of parasite clearance time by t test with Mann-Whitney test revealed significantly faster parasite clearance time for the ASAP treatment vs AP in Eastern Cambodia only (P = .04). There were no significant differences in parasite clearance half-life overall. The median PCT1/2 was significantly higher at 5.34 nM for parasites with the K13 propeller C580Y mutation vs 3.42 for parasites without C580Y (P = .0032).
Figure 4.
Figure 4.
Immediate ex vivo Plasmodium falciparum (P.f.) parasite resistance. A, Overall ex vivo P.f. parasite sensitivity to commonly used antimalarial drugs in 2014 from field surveillance stations in Cambodia. Red bars represent geometric mean values, whereas green dashed lines represent values for the W2 Indochina P.f. clone. B. Ex vivo parasite sensitivity by histidine-rich protein 2 enzyme-linked immunosorbent assay to atovaquone and cycloguanil for evaluable isolates on the day of study screening before treatment (D0) and day of recrudescence (DR). Baseline assays were interpretable for only 93 of 202 isolates for atovaquone (46%) and 46 of 160 (29%) for cycloguanil. Clear dots represent parasites with the cytb atovaquone resistance gene wild-type, and blue dots represent K13 propeller status unknown. Red bars in each column represent geometric mean 50% inhibitory concentration (IC50) values, whereas dashed green lines represent mean values for the chloroquine-resistant W2 Indochina clone run simultaneously for each assay. Dashed blue lines represent geometric mean values for the atovaquone-resistant C2B clone. Dashed orange lines represent geometric mean values for the chloroquine-sensitive D6 clone. Geometric mean atovaquone IC50 for parasites with the C580Y mutation was significantly higher (4.57 nM; n = 94) than for non-C580Y parasites (1.29 nM; n = 4; P = .001).
Figure 5.
Figure 5.
Gametocyte carriage by light microscopy. A, Number and percentage of subjects found to have Plasmodium falciparum gametocytes by light microscopy from each treatment arm during 6 weeks of follow-up. B and C, Median parasite gametocyte densities over the first 2 weeks of follow-up for those subjects who were originally gametocytemic, normalized to baseline values on D0 (100%).
Figure 6.
Figure 6.
Atovaquone levels after drug administration. A, Atovaquone levels in nanomoles (nM) per milliliter (mL) of plasma in patients with uncomplicated malaria dosed with atovaquone-proguanil (AP) or artesunate-atovaquone-proguanil (ASAP) for 3 days. Horizontal lines represent mean parasite atovaquone 50% inhibitory concentration (IC50) values in nM at baseline for the respective treatment groups. Dashed horizontal lines represent mean parasite atovaquone IC50 values at baseline for the respective study groups. B, Atovaquone levels in patients who were cured (ACPR) and those who had a malaria recurrence. Horizontal lines represent mean parasite atovaquone IC50 values in nM at baseline for the respective groups. C, Median day 7 drug levels of atovaquone for ACPR and recrudescent patients. One patient in the recrudescence group missed follow-up on day 7.

Source: PubMed

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