William M. Watkins

Averting a malaria disaster

Estimates for the annual mortality from malaria range from 0·5 to 2·5 million deaths. The burden of this enormous toll, and the concomitant morbidity, is borne by the world’s poorest countries. Malaria morbidity and mortality have been held in check by the widespread availability of cheap and effective antimalarial drugs. The loss of these drugs to resistance may represent the single most important threat to the health of people in tropical countries. Chloroquine has been the mainstay of antimalarial drug treatment for the past 40 years, but resistance is now widespread and few countries are u n a f f e c t e d . 1 Pyrimethamine-sulphadoxine (PSD) is usually deployed as a successor to chloroquine. Both these antimalarials cost less than US

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

0.20 per adult treatment course, but the drugs required to treat multidrug-resistant falciparum malaria (quinine, mefloquine, halofantrine) are over ten times more expensive and cannot be afforded by most tropical countries— especially those in Africa, where it is estimated that more than 90% of the world’s malaria deaths occur. Resistance to chloroquine is widespread across Africa and resistance to PSD is increasing. 2 A health calamity looms within the next few years. 3 As treatments lose their effectiveness, morbidity and mortality from malaria will inevitably continue to rise. Can this disaster be prevented? Can we really “roll back malaria”, as the new Director-General of WHO has demanded? 4

Pyrimethamine–sulfadoxine resistance in Plasmodium falciparum: what next?

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.

Life-threatening bacteraemia in HIV-1 seropositive adults admitted to hospital in Nairobi, Kenya

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.

Resistance to antifolates in Plasmodium falciparum monitored by sequence analysis of dihydropteroate synthetase and dihydrofolate reductase alleles in a large number of field samples of diverse origins

During 6 months, 506 consecutive adult emergency admissions to hospital in Nairobi were enrolled in a study of bacteraemia and HIV infection. 19% were HIV-1 antibody positive. Significantly more HIV-seropositive than seronegative patients had bacteraemia (26% vs 6%). The predominant organisms isolated from the seropositive patients were Salmonella typhimurium and Streptococcus pneumoniae. Mortality was higher in the seropositive than in the seronegative bacteraemic patients. The findings suggest that non-opportunistic bacteria are important causes of morbidity and mortality in HIV-infected individuals in Africa.

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

Resistance of Plasmodium falciparum to antifolate chemotherapy is a significant problem where combinations such as Fansidar (pyrimethamine-sulfadoxine; PYR-SDX) are used in the treatment of chloroquine-resistant malaria. Antifolate resistance has been associated with variant sequences of dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS), the targets of PYR and SDX respectively. However, while the nature and distribution of mutations in the dhfr gene are well established, this is not yet the case for dhps. We have thus examined by DNA sequence analysis 141 field samples from several geographical regions with differing Fansidar usage (West and East Africa, the Middle East and Viet Nam) to establish a database of the frequency and repertoire of dhps mutations, which were found in 60% of the samples. We have also simultaneously determined from all samples their dhfr sequences, to better understand the relationship of both types of mutation to Fansidar resistance. Whilst the distribution of mutations was quite different across the regions surveyed, it broadly mirrored our understanding of relative Fansidar usage. In samples taken from individual patients before and after drug treatment, we found an association between the more highly mutated forms of dhps and/or dhfr and parasites that were not cleared by antifolate therapy. We also report a novel mutation in a Pakistani sample at position 16 of DHFR (A16S) that is combined with the familiar C59R mutation, but is wild-type at position 108. This is the first observation in a field sample of a mutant dhfr allele where the 108 codon is unchanged.

Treatment of Plasmodium falciparum malaria with pyrimethamine-sulfadoxine: selective pressure for resistance is a function of long elimination half-life

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.

WHO, the Global Fund, and medical malpractice in malaria treatment

In an area of continuing transmission of Plasmodium falciparum on the Kenya coast, children treated with pyrimethamine-sulfadoxine experienced rapid parasite clearance, although a high proportion became reinfected within a short time. The frequency of pyrimethamine resistance in vitro in new infections was higher during the elimination phase of drug from a previous treatment. In infections which occurred at times when predicted residual drug concentrations were no longer inhibitory, incidence of pyrimethamine resistance was no different from the natural or background frequency. These results are discussed in terms of the selective pressure for resistance which is exerted by drugs with long elimination half-lives and a consideration of possible ways by which the problem might be addressed.

Changing home treatment of childhood fevers by training shop keepers in rural Kenya.

Amir Attaran and colleagues highlight a very serious public-health issue. Provision of ineffective drugs for a life-threatening disease is indefensible. There is no doubt that chloroquine is now ineffective for the treatment of falciparum malaria in nearly all tropical countries and that its usual successor sulfadoxine-pyrimethamine is falling fast to resistance. As a result malaria mortality in eastern and southern Africa where hundreds of thousands of children die each year from the infection has doubled in the past decade. We have failed to roll back malaria and we in the developed world bear the responsibility for this humanitarian disaster. Malaria is not an insoluble problem. We already have the tools (insecticides bednets highly effective drugs) to reduce substantially the terrible death toll. But we are not providing them to the people who need them desperately but who cannot pay for them. Only a tiny fraction of the millions with malaria today receive highly effective treatments. The donors must take some responsibility for this failure. Given the choice between receiving donor support for ineffective chloroquine or sulfadoxine-pyrimethamine and receiving nothing most countries have naturally opted for the former. It is not easy to protest particularly when the main donors and the representatives of international organisations both claim these drugs are still “programmatically effective”. (excerpt)

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

Summary background Malaria control in Africa relies primarily on early effective treatment for clinical disease, but most early treatments for fever occur through self‐medication with shop‐bought drugs. Lack of information to community members on over‐the‐counter drug use has led to widespread ineffective treatment of fevers, increased risks of drug toxicity and accelerating drug resistance. We examined the feasibility and measured the likely impact of training shop keepers in rural Africa on community drug use.