Atovaquone-proguanil (ATQ-PRO, Malarone, GlaxoSmithKline Inc., Mississauga, ON, Canada) is a common first-line agent for prophylaxis and therapy for malaria caused by Plasmodium falciparum, because of its efficacy, convenient dosing schedule, and favorable side effect profile. Since 2002 there have been several case reports of ATQ-PRO treatment failure, believed to result from acquired mutations in plasmodial cytochrome b.1–8 We present the first North American pediatric case of genetically confirmed ATQ-PRO failure and discuss the clinical implications of this and similar published cases.
In the spring of 2006, a 3 1/2-year-old boy presented to the Emergency Room at the Children's Hospital of Eastern Ontario with a 7-day history of fever, vomiting, and decreased oral intake. He had no neurologic symptoms, decreased level of consciousness, icterus or abdominal pain. The patient had arrived in Canada 7 days earlier from a refugee camp in Mozambique, where he had been born and raised.
Past medical history was significant for 3 prior episodes of presumptive malaria, for which he was treated and recovered uneventfully. The patient's parents do not recall which antimalarial therapy he had received, and medical records from Mozambique are not available. He was otherwise healthy with no clinical or laboratory evidence of failure to thrive, human immunodeficiency virus-1 or Mycobacterium tuberculosis infection. He received no regular medications and had no allergies.
On examination, he had a temperature of 39.5°C, pulse of 168 beats/min, respiratory rate of 30 breaths/min, blood pressure of 104/66 mm Hg, and he weighed 13.3 kgs (25th percentile). Notable findings were a 2 over 6 systolic ejection murmur, a palpable liver edge, and a spleen palpable 1 cm below the costal margin.
Initial blood work showed a normocytic anemia (hemoglobin 72 g/L) with normal white blood cell count and platelet count. Lactate dehydrogenase was elevated at 1261 U/L, but other chemistries were within normal limits, and there was no indication of intravascular hemolysis. Malaria smears were positive for Plasmodium falciparum malaria with a parasitemia of 1.2%.
He was given a first dose of ATQ-PRO (250 mg/100 mg) within 1 hour of diagnosis and admitted to hospital. He had no further vomiting or diarrhea. Fever persisted during the first 36 hours of the admission. By the end of the second day the patient was afebrile and clinically well. Parasitemia initially increased to 2.8% in the first 15 hours and then declined in the second (2.3%), third (1.6%), and fourth (0.8%) days of hospitalization, and was undetectable on the fifth day. Because of a slower than expected resolution of parasitemia, the patient received a 5-day course of ATQ-PRO, and was discharged home with outpatient follow-up.
At the 1-week follow-up, parents reported that he was well and afebrile. The peripheral blood smear showed only 2 gametocytes. At the 4-week outpatient assessment, the patient appeared well with a normal physical examination; however, blood smears demonstrated asexual forms of P. falciparum with a parasitemia of 3.2%.
When reassessed in emergency room 4 hours later, he had developed a fever of 38.1°C axillary. Clinical examination was unremarkable. Because of recrudescent parasitemia, the possibility of ATQ-PRO treatment failure was considered, and he was given intravenous quinine and clindamycin. Five hours after his initial parenteral quinine dose, the parasitemia had decreased to 0.3%, and was undetectable by 12 hours. On the second day of admission, the patient remained afebrile with undetectable parasitemia, and therapy was changed to oral quinidine and clindamycin (suspensions) to complete 5 further days of therapy. At 1 and 4 weeks after his retreatment course he was feeling well, and had no detectable parasitemia.
Before initiating quinine treatment, a blood sample was sent to a reference laboratory for polymerase chain reaction amplification and genetic analysis to detect parasite mutations previously associated with ATQ-PRO resistance. Plasmodial DNA was extracted from the blood sample collected from the recurrent malarial illness using Qiagen columns (Qiagen, Chatsworth, CA). The cytochrome b gene was amplified by polymerase chain reaction and sequenced from the recrudescent falciparum isolate to detect mutations associated with resistance to atovaquone as described.3,6 Sequence analysis of the recrudescent isolate revealed a mutation at position 268 specifying a change from tyrosine to serine. This Tyr268Ser mutation has been previously associated with clinical failure and a 9364-fold increase in the 50% inhibitory concentration of atovaquone.9
Monotherapy with either ATQ or PRO is of limited efficacy. The mechanism of action of ATQ is inhibition of the electron transport chain at cytochrome bc1 and collapse of electropotential across the mitochondrial membrane.10 Monotherapy rapidly selects for resistance mutations in plasmodial cytochrome b, which lead to treatment failure in 33% of cases.9 Similarly, resistance to PRO, which inhibits plasmodial dihydrofolate reductase (dhfr) via its metabolite cycloguanil, is rapidly engendered by PRO monotherapy and is widespread.9 Conversely, the combination ATQ-PRO is highly effective for chemoprophylaxis and therapy for malaria and treatment failure is rare.
Our case is the first reported clinical failure of ATQ-PRO therapy in a child in North America. In postmarketing reporting we have identified 13 previous cases of ATQ-PRO treatment failure associated with single mutations at codon 268 (substituting serine, cysteine, or asparagine for tyrosine) in plasmodial cytochrome b.1–8 (Table 1) There has also been one case of clinical success despite a serine substitution for tyrosine at codon 268,2 and several cases of clinical treatment failure not associated with any known cytochrome b mutations.2,5,7
Clinical failure may result from severity of initial disease, problems with drug dosage or administration, drug malabsorption, or from antimalarial resistance in the parasite. In 14 published cases of relapsed malaria associated with single mutations at codon 268 (including our own), there was 1 case of severe parasitemia, and 1 case wherein the dose of ATQ-PRO was considered inadequate. Therefore, in 12 cases there is no explanation for resistance other than mutation in cytochrome b.
Reports in which parasite genotypes are analyzed from samples before and after therapy with ATQ-PRO suggest that resistance mutations in cytochrome b are induced by therapy.3,6–8 Less frequently, a population of preexisting cytochrome b mutants may be selected by therapy.2 Of 10 cases of ATQ-PRO treatment failure for which pretherapy P. falciparum isolates were analyzed, only 1 bore a resistance mutation in cytochrome b on day 0.2 Similarly, a survey of 477 pretherapy P. falciparum isolates from travelers returning from Africa and the Indian Ocean revealed no occurrence of cytochrome b resistance mutation.11
Thus, it seems unlikely our patient's pretherapy P. falciparum bore the Tyr268Ser mutation, although a sample of the parasite from day 0 was not available. It is also unlikely that he had ever received ATQ-PRO before the initial malaria presentation to our institution, as the Office of the United Nations’ High Commissioner for Refugees, which runs the camp where he lived, does not use this drug.12
We believe it more likely that the failure to clear parasitemia as expected before the fifth day of initial ATQ/PRO therapy may have resulted from rapidly induced Tyr268Ser mutation. Analysis of sequential isolates from the 6 cases reported by Musset et al7 supports the concept of induction of near-identical cytochrome b codon 268 mutations during therapy by varied pathways in a process of parallel evolution.13 Regardless of the provenance of Tyr268Ser mutation in our patient's case, the need for prolongation of therapy to 5 days retrospectively seems to have been a predictor of subsequent recrudescence.
In summary, this is the first pediatric case of ATQ-PRO resistance in North America. The case highlights the following 2 points that can directly impact the outcome of disease: (1) failure to defervesce and clear Plasmodium parasitemia in an appropriate timeframe may indicate the presence of antimalarial resistance; and (2) follow-up assessments at 1 and 4 weeks posttherapy, including repeat malaria smears, are integral parts of proper management of malaria.
1.Fivelman QL, Butcher GA Adagu IS, et al. Malarone
treatment failure in in vitro
confirmation of resistance
of Plasmodium falciparum
isolate from Lagos, Nigeria. Malaria J.
2.Färnert A, Lindberg J, Gil P, et al. Evidence of Plasmodium falciparum
malaria resistant to atovaquone and proguanil hydrochloride: case reports. BMJ
3.Schwartz E, Bujanover S, Kain KC. Genetic confirmation of atovaquone-proguanil
-resistant Plasmodium falciparum
malaria acquired by a nonimmune traveler to East Africa. Clin Infect Dis
4.David KP, Alifrangis M, Salanti A, et al. Atovaquone/proguanil resistance
in Africa: a case report. Scand J Infect Dis
5.Wichmann O, Muehlberger N, Jelinek T, et al. Screening for mutations related to atovaquone/proguanil resistance
in treatment failures and other imported isolates of Plasmodium falciparum
. J Infect Dis
6.Kuhn S, Gill MJ, Kain KC. Emergence of atovaquone-proguanil resistance
during treatment of Plasmodium falciparum
malaria acquired by a non-immune North American traveler to West Africa. Am J Trop Med Hyg
7.Musset L, Bouchaud O, Matheron S, et al. Clinical atovaquone-proguanil resistance
of Plasmodium falciparum
associated with cytochrome b codon 268 mutations. Microbes Infect
8.Legrand E, Demar M, Volney B, et al. First case of emergence of atovaquone resistance
in Plasmodium falciparum
during second-line atovaquone-proguanil
treatment in South America. Antimicrob Agents Chemother
9.Looaresuwan S, Viravan C, Webster HK, et al. Clinical studies of atovaquone, alone or in combination with other antimalarial drugs, for treatment of acute uncomplicated malaria in Thailand. Am J Trop Med Hyg
10.Srivastava IK, Vaidya AB. A mechanism for the synergistic antimalarial action of atovaquone and proguanil. Antimicrob Agents Chemother
11.Musset L, Pradines B, Parzy D, et al. Apparent absence of atovaquone/proguanil resistance
in 477 Plasmodium falciparum
isolates from untreated French travellers. J Antimicrob Chemother
12.United Nation High Commissioner for Refugees. UNHCR Strategic Plan for Malaria Control 2005–2007
. Geneva, Switzerland: United Nation High Commissioner for Refugees; 2006.
13.Musset L, Le Bras J, Clain J. Parallel evolution of adaptive mutations in Plasmodium falciparum
mitochondrial DNA during atovaquone-proguanil
treatment. Mol Biol Evol