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Trans R Soc Trop Med Hyg. Author manuscript; available in PMC 2009 August 3.
Published in final edited form as:
PMCID: PMC2720017
EMSID: UKMS27452

Antifolate resistance in Africa and the 164-dollar question

Summary

The spread of Plasmodium falciparum carrying a quadruply mutated dhfr gene to Africa has been widely predicted to have profoundly adverse consequences, as such parasites in vitro are highly resistant to antifolate inhibitiors, still a mainstay of antimalarial drug regimes in this region. Studies of parasites from Southeast Asia demonstrate a strong connection between the I164L-bearing quadruple mutant form and failure of sulfadoxine—pyrimethamine (SP) treatment. However, a recent study reported in this issue of Transactions documents the low-level incidence in an area of Kenya of quadruply mutant parasites which, in the majority of cases, appear to have been cleared by a standard SP treatment regime, contrary to expectations.

Keywords: Malaria, Plasmodium falciparum, Dihydrofolate reductase, Quadruple mutants, Drug treatment, Sulfadoxine-pyrimethamine

Despite the recent adoption of artemisinin (or derivative) combination therapy (ACT) as a first-line antimalarial regime by many African countries, delays in actual deployment and cost implications mean that the use of the cheap antifolate combination sulfadoxine—pyrimethamine (SP) is still widespread, albeit decreasingly efficacious. Moreover, SP is still promoted by WHO as the safest option for preventing malaria during pregnancy. Resistance of Plasmodium falciparum to SP is caused by well documented mutations in the dhps and dhfr genes that encode the enzymes in the folate pathway that are the targets of these drugs. The most common genotype found in areas of high SP resistance in Africa are triply mutant versions of dhfr (usually S108N, N51I and C59R, at the amino acid level), together with doubly mutant versions of dhps (usually A437G and K540E). However, in Southeast Asia, where so much drug resistance appears to originate, a quadruply mutant form of dhfr that includes an I164L alteration has been increasingly observed over the years, after initial identification in the late 1980s. In the laboratory, this form of the enzyme binds pyrimethamine some 600 times less tightly than the wild type and about seven times less than the triply mutant form (Sirawaraporn et al., 1997); thus parasites carrying such a quadruple combination of mutations are refractory in vitro to SP concentrations higher than those achievable in the human host in vivo. Moreover, such parasites are also resistant to the more potent antifolate combination of chlorproguanil—dapsone (LapDap), only recently licensed and introduced into some parts of Africa. It was not surprising then that many commentators predicted disastrous consequences when such a highly mutant form of P. falciparum arose in or migrated to Africa, where transmission rates and mortality are so much higher than in Southeast Asia (e.g. Wichmann et al., 2003). Inevitably, such parasites have indeed been detected recently in several countries in East Africa, with the I164L alteration added to a background of either double (S108N, N51I or S108N, C59R) or triple (S108N, N51I, C59R) mutations. At least one haplotype of such parasites has been shown by analysis of microsatellite markers to derive from a strain first detected in Thailand (Maiga et al., 2007). In anticipation of this spread, diagnostic tests for I164L had already been included in PCR assays in the more recent surveillance programmes in Africa and potential new inhibitors able to bind strongly to the quadruply mutant form of DHFR have been under investigation for some time (e.g. Hankins et al., 2001; Kamchonwongpaisan et al., 2005).

In a recent study of 48 adult Thai patients (Krudsood et al., 2005), the I164L mutation was reported as the ‘main determinant’ of treatment failure. Even when antifolates were combined with artesunate in a proguanil/dapsone/artesunate triple combination, this was found insufficient to clear more than 73% of infections by parasites carrying this mutation in addition to those at positions 51, 59 and 108. However, in vitro kinetic studies have also suggested that this quadruply mutant version of DHFR is a highly compromised enzyme (Cortese and Plowe, 1998; Sirawaraporn et al., 1997) and so parasites depending upon such an enzyme might suffer significant fitness costs. This could in principle retard the spread of such parasites, such that the deployment in Africa of ACT-type therapies based entirely on non-antifolate inhibitors could be well established before a complete loss of antifolate efficacy occurred. Moreover, there is now some evidence from the paper by Hamel et al. (this issue) published in this issue of Transactions that the dire consequences envisaged when the quadruply mutant dhfr form appeared in Africa may have been unduly pessimistic. In this study, conducted in an area of intense perennial transmission in Kenya, the incidence of I164L was monitored, together with associated SP failure rates [for a detailed description of the molecular analyses, see McCollum et al. (2006)]. Out of nearly 500 cases where parasitaemia was detected either by microscopy or PCR, the I164L mutation was found in 14 patients, of which seven carried parasites detectable by microscopy at day 0. Although in only four of these I164L was found unambiguously in the quadruply mutant dhfr type, three of those four cases were cleared of parasites at day 14 after SP treatment (including in one HIV-positive patient with a low CD4+ cell count). Moreover, in the one treatment failure, the quadruple mutant was unexpectedly absent in the day 7 parasites. Of the other seven patients, who had very low parasitaemias only detectable by PCR, only two were unambiguously carrying the quadruply mutant type, neither of which went on to develop parasitaemias where I164L was evident during the subsequent six months of the study.

The authors conclude that there is no clear association between the incidence of I164L and SP failure in their study, while conceding that these are very small sample numbers from a population of (non-pregnant) adults. However, the results do suggest that the situation with quadruply mutant parasites may not be as simple as had been assumed and that context is likely to be very important. Could it be, for example, that the parasites monitored by Krudsood et al. (2005) in Thailand are also carrying as yet unknown determinants enhancing resistance that are absent from the populations studied in Kenya? This possibility is perhaps indicated by the fact that artesunate in combination with the antifolates was also ineffective against the Thai I164L parasites in nearly 30% of cases. Moreover, the latter parasites additionally carried multiple mutations in dhps, whereas the status of this gene was not monitored in the Kenyan samples. Clearly, the degree to which highly mutant parasites will contribute to drug failure depends upon a number of factors, including the density of such parasites in the inoculum, the fitness of these parasites relative to less mutated forms that might also be present during the early reproductive stages, and the level of acquired immunity in the host. With regard to the last, levels of partial immunity are generally higher in Africa than in Southeast Asia. However, even if a proportion of African adults are able to clear such parasites the likelihood is that children and pregnant women, with their lower levels of immunity, will be unable to do this and, as yet, there are essentially no data pertaining to these groups. So, returning to the question of whether the presence of the I164L change in quadruply mutant dhfr will inevitably lead to antifolate treatment failure, as was widely taken for granted, the answer appears to be no. However, the extent of the exceptions to this ‘rule’ is far from clear at the present time with the very limited data to hand and it is important that a more comprehensive picture be obtained as quickly as possible, so that drug treatment strategies can be tailored accordingly.

Acknowledgments

Funding: Wellcome Trust grant 073896.

Footnotes

Conflicts of interest: None declared.

Ethical approval: Not required.

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