Peter Winstanley

Mutations in Plasmodium falciparum Dihydrofolate Reductase and Dihydropteroate Synthase and Epidemiologic Patterns of Pyrimethamine-Sulfadoxine Use and Resistance

To assess the relationship between mutations in Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) and clinical pyrimethamine-sulfadoxine resistance, polymerase chain reaction surveys and analyses for new mutations were conducted in four countries with increasing levels of pyrimethamine-sulfadoxine resistance: Mali, Kenya, Malawi, and Bolivia. Prevalence of mutations at DHFR codon 108 and a new mutation at DHPS 540 correlated with increased pyrimethamine-sulfadoxine resistance (P < .05). Mutations at DHFR 51, DHFR 59, and DHPS 437 correlated with resistance without achieving statistical significance. Mutations at DHFR 164 and DHPS 581 were common in Bolivia, where pyrimethamine-sulfadoxine resistance is widespread, but absent in African sites. Two new DHFR mutations, a point mutation at codon 50 and an insert at codon 30, were found only in Bolivia. DHFR and DHPS mutations occur in a progressive, stepwise fashion. Identification of specific sets of mutations causing in vivo drug failure may lead to the development of molecular surveillance methods for pyrimethamine-sulfadoxine resistance.

Pyrimethamine–sulfadoxine resistance in Plasmodium falciparum: what next?

Chemotherapy remains the only practicable tool to control falciparum malaria in sub-Saharan Africa, where >90% of the worlds burden of malaria mortality and morbidity occurs. Resistance is rapidly eroding the efficacy of chloroquine, and the combination pyrimethamine-sulfadoxine is the most commonly chosen alternative. Resistant populations of Plasmodium falciparum were selected extremely rapidly in Southeast Asia and South America. If this happens in sub-Saharan Africa, it will be a public health disaster because no inexpensive alternative is currently available. This article reviews the molecular mechanisms of this resistance and discusses how to extend the therapeutic life of antifolate drugs.

Childhood deaths in Africa: uses and limitations of verbal autopsies

The verbal autopsy (VA) is an epidemiological tool that is widely used to ascribe causes of death by interviewing bereaved relatives of children who were not under medical supervision at the time of death. This technique was assessed by comparison with a prospective survey of 303 childhood deaths at a district hospital in Kenya where medically confirmed diagnoses were available. Common causes of death were detected by VA with specificities greater than 80%. Sensitivity of the VA technique was greater than 75% for measles, neonatal tetanus, malnutrition, and trauma-related deaths; however, malaria, anaemia, acute respiratory-tract infection, gastroenteritis, and meningitis were detected with sensitivities of less than 50%. There may have been unwarranted optimism in the ability of VAs to detect some of the major causes of death, such as malaria, in African children. VA used in malaria-specific intervention trials should be interpreted with caution and only in the light of known sensitivities and specificities.

Towards an understanding of the mechanism of pyrimethamine-sulfadoxine resistance in Plasmodium falciparum: genotyping of dihydrofolate reductase and dihydropteroate synthase of Kenyan parasites.

ABSTRACT The antifolate combination of pyrimethamine (PM) and sulfadoxine (SD) is the last affordable drug combination available for wide-scale treatment of falciparum malaria in Africa. Wherever this combination has been used, drug-resistant parasites have been selected rapidly. A study of PM-SD effectiveness carried out between 1997 and 1999 at Kilifi on the Kenyan coast has shown the emergence of RI and RII resistance to PM-SD (residual parasitemia 7 days after treatment) in 39 out of 240 (16.25%) patients. To understand the mechanism that underlies resistance to PM-SD, we have analyzed the dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genotypes of 81 patients. Fifty-one samples were obtained, before treatment, from patients who remained parasite free for at least 7 days after treatment. For a further 20 patients, samples were obtained before treatment and again when they returned to the clinic with parasites 7 days after PM-SD treatment. Ten additional isolates were obtained from patients who were parasitemic 7 days after treatment but who were not sampled before treatment. More than 65% of the isolates (30 of 46) in the initial group had wild-type or double mutant DHFR alleles, and all but 7 of the 47 (85%) had wild-type DHPS alleles. In the paired (before and after treatment) samples, the predominant combinations of DHFR and DHPS alleles before treatment were of triple mutant DHFR and double mutant DHPS (41% [7 of 17]) and of double mutant DHFR and double mutant DHPS (29% [5 of 17]). All except one of the posttreatment isolates had triple mutations in DHFR, and most of these were “pure” triple mutants. In these isolates, the combination of a triple mutant DHFR and wild-type DHPS was detected in 6 of 29 cases (20.7%), the combination of a triple mutant DHFR and a single mutant (A437G) DHPS was detected in 4 of 29 cases (13.8%), and the combination of a triple mutant DHFR and a double mutant (A437G, L540E) DHPS was detected in 16 of 29 cases (55.2%). These results demonstrate that the triply mutated allele of DHFR with or without mutant DHPS alleles is associated with RI and RII resistance to PM-SD. The prevalence of the triple mutant DHFR-double mutant DHPS combination may be an operationally useful marker for predicting the effectiveness of PM-SD as a new malaria treatment.

FREQUENCY OF SEVERE NEUTROPENIA ASSOCIATED WITH AMODIAQUINE PROPHYLAXIS AGAINST MALARIA

6 out of 7 patients with severe neutropenia associated with the use of amodiaquine for malaria prophylaxis amodiaquine (400 mg weekly) plus proguanil (200 mg daily); 1 of these patients had also taken cotrimoxazole and another had taken sulphaguanidine. The 7th patient had taken amodiaquine alone, but at a higher dose. A retrospective analysis suggests that the frequency of severe neutropenia complicating amodiaquine taken prophylactically may be as high as 1 in 2000.

Chlorproguanil-dapsone for treatment of drug-resistant falciparum malaria in Tanzania.

BACKGROUND Resistance to the affordable malaria treatments chloroquine and pyrimethamine-sulfadoxine is seriously impeding malaria control through treatment in east Africa. We did an open, alternate drug allocation study to assess the efficacy of chlorproguanil-dapsone in the treatment of falciparum malaria clinically resistant to pyrimethamine-sulfadoxine. METHODS Children younger than 5 years with non-severe falciparum malaria, attending Muheza district hospital in Tanzania, were treated with the standard regimen of pyrimethamine-sulfadoxine. Patients whose clinical symptoms resolved but who remained parasitaemic 7 days after pyrimethamine-sulfadoxine were followed up for 1 month. Clinical malaria episodes were retreated with either single dose pyrimethamine-sulfadoxine or a 3-day regimen of chlorproguanil-dapsone. Those with parasitaemia after 7 days were treated with chlorproguanil-dapsone. Parasite DNA was collected on day 7 after first treatment with pyrimethamine-sulfadoxine and we looked for point mutations in the genes encoding dihydrofolate reductase (dhfr) and dyhydropteroate synthetase (dhps). FINDINGS 360 children were enrolled and treated with pyrimethamine-sulfadoxine. On day 7, 192 (55%) of 348 had cleared parasitaemia. Of the remaining 156 parasitaemic children, 140 (90%) were followed up to day 28, and 92 (66%) of 140 developed clinical malaria. These 92 patients were alternately retreated with either pyrimethamine-sulfadoxine (46) or chlorproguanil-dapsone (46). 28 (61%) of 46 children retreated with pyrimethamine-sulfadoxine were still parasitaemic at day 7, compared with three (7%) of 44 [corrected] children retreated with chlorproguanil-dapsone. Resistance to pyrimethamine-sulfadoxine increased from 45% (156/348) at the first treatment to 61% (28/46) after retreatment. 83 of 85 parasite isolates collected after the first pyrimethamine-sulfadoxine treatment, and before and after the second treatments with pyrimethamine-sulfadoxine and chlorproguanil-dapsone showed triple-mutant dhfr alleles, associated with a variety of dhps mutations. INTERPRETATION Most patients treated with pyrimethamine-sulfadoxine, who remain parasitaemic at day 7, develop new malaria symptoms within 1 month. Chlorproguanil-dapsone was a practicable therapy under these circumstances. Analysis of parasite dhfr and dhps before and after treatment supports the view that pyrimethamine-sulfadoxine resistance in this part of Africa is primarily due to parasites with three mutations in the dhfr domain.

Efficacy and safety of intermittent preventive treatment with sulfadoxine-pyrimethamine for malaria in African infants: a pooled analysis of six randomised, placebo-controlled trials

BACKGROUND Intermittent preventive treatment (IPT) is a promising strategy for malaria control in infants. We undertook a pooled analysis of the safety and efficacy of IPT in infants (IPTi) with sulfadoxine-pyrimethamine in Africa. METHODS We pooled data from six double-blind, randomised, placebo-controlled trials (undertaken one each in Tanzania, Mozambique, and Gabon, and three in Ghana) that assessed the efficacy of IPTi with sulfadoxine-pyrimethamine. In all trials, IPTi or placebo was given to infants at the time of routine vaccinations delivered by WHOs Expanded Program on Immunization. Data from the trials for incidence of clinical malaria, risk of anaemia (packed-cell volume <25% or haemoglobin <80 g/L), and incidence of hospital admissions and adverse events in infants up to 12 months of age were reanalysed by use of standard outcome definitions and time periods. Analysis was by modified intention to treat, including all infants who received at least one dose of IPTi or placebo. FINDINGS The six trials provided data for 7930 infants (IPTi, n=3958; placebo, n=3972). IPTi had a protective efficacy of 30.3% (95% CI 19.8-39.4, p<0.0001) against clinical malaria, 21.3% (8.2-32.5, p=0.002) against the risk of anaemia, 38.1% (12.5-56.2, p=0.007) against hospital admissions associated with malaria parasitaemia, and 22.9% (10.0-34.0, p=0.001) against all-cause hospital admissions. There were 56 deaths in the IPTi group compared with 53 in the placebo group (rate ratio 1.05, 95% CI 0.72-1.54, p=0.79). One death, judged as possibly related to IPTi because it occurred 19 days after a treatment dose, was subsequently attributed to probable sepsis. Four of 676 non-fatal hospital admissions in the IPTi group were deemed related to study treatment compared with five of 860 in the placebo group. None of three serious dermatological adverse events in the IPTi group were judged related to study treatment compared with one of 13 in the placebo group. INTERPRETATION IPTi with sulfadoxine-pyrimethamine was safe and efficacious across a range of malaria transmission settings, suggesting that this intervention is a useful contribution to malaria control. FUNDING Bill & Melinda Gates Foundation.

Chemotherapy for Falciparum Malaria: The Armoury, the Problems and the Prospects

Peter Winstanley here describes the pharmacology and therapeutics of the main drugs used for falciparum malaria in the tropical setting, rather than in the developed world, as an overview for newcomers to the field. He then examines some of the current major problems and prospects for the future.

Severe anaemia in children living in a malaria endemic area of Kenya

Severe anaemia is an important cause of morbidity and mortality in African children, but the causes, particularly falciparum malaria, are difficult to determine. We assessed the contribution of falciparum malaria to anaemia in Kenyan children by clinical examination and measurement of parasitaemia and haemoglobin (Hb) concentration in 559 children in the community and in 2412 children admitted to Kilifi district hospital during a 2‐year period. We also attempted to characterize severe malarial anaemia by examining the causes and pathophysiology of anaemia in 101 children admitted with Hb concentration 50 g/l during a 1‐year period. Plasmodium falciparum infection was associated with reduced Hb concentration in children in the community and in those admitted to hospital irrespective of diagnosis. Falciparum malaria was the primary cause in 46 cases (46%) of severe anaemia admitted to hospital. There was no difference in the frequency of haemolysis or dyserythropoiesis in the children with malarial anaemia and those with anaemia from other causes, such as iron deficiency or sickle cell disease. The mortality rate in the children with severe malarial anaemia was 8.6% compared with 3.6% in children with severe anaemia due to other causes. Falciparum malaria does not present with a characteristic clinical or haematological picture, but is a major cause of the morbidity and mortality in children with severe anaemia who live on the Kenyan coast, a malaria endemic area.

The efficacy of antifolate antimalarial combinations in Africa: a predictive model based on pharmacodynamic and pharmacokinetic analyses

At present, effective treatment for non-severe malaria is the most important malaria control strategy in Africa. Pyrimethamine-sulfadoxine (PSD) is rapidly becoming the first-line treatment in areas of chloroquine resistance, although the parasite chemoresistance factors that dispose towards clinical failure with PSD are still unclear. Here, Bill Watkins and colleagues analyse the relationship between the pharmacokinetic properties of two treatment combinations (PSD and chlorproguanil-dapsone) in vivo and the respective in vitro isobolograms for parasites with specific drug-resistance patterns. From this relationship, they develop a hypothesis that may explain clinical drug failure and differential efficacy between treatments. The deductions can be tested in field studies to validate or refute the model.