Malaria genomics meets drug-resistance phenotyping in the field

A report of the 2nd Wellcome Trust Conference on Genomic Epidemiology of Malaria, Hinxton, UK, 14-17 June 2009

Drug resistance maps to guide intermittent preventive treatment of malaria in African infants

Intermittent preventive treatment of infants (IPTi) with sulphadoxine pyrimethamine (SP) is recommended as an additional malaria control intervention in high transmission areas of sub-Saharan Africa, provided its protective efficacy is not compromised by SP resistance. A significant obstacle in implementing SP-IPTi, is in establishing the degree of resistance in an area. Since SP monotherapy is discontinued, no contemporary measures of in vivo efficacy can be made, so the World Health Organisation has recommended a cut-off based upon molecular markers, stating that SP-IPTi should not be implemented when the prevalence of the dhps 540E mutation among infections exceeds 50%. We created a geo-referenced database of SP resistance markers in Africa from published literature. By selecting surveys of malaria infected blood samples conducted since 2004 we have mapped the contemporary prevalence of dhps 540E. Additional maps are freely available in interactive form at http://www.drugresistancemaps.org/ipti/. Eight countries in East Africa are classified as unsuitable for SP-IPTi when data are considered at a national level. Fourteen countries in Central and West Africa were classified as suitable while seven countries had no available contemporary data to guide policy. There are clear deficiencies in molecular surveillance data coverage. We discuss requirements for ongoing surveillance of SP resistance markers in support of the use of SP-IPTi

Unexpected selections of Plasmodium falciparum polymorphisms in previously treatment-naïve areas after monthly presumptive administration of three different anti-malarial drugs in Liberia 1976-78.

BACKGROUND: To assess the effect on malaria prevalence, village specific monthly administrations of pyrimethamine, chlorproguanil, chloroquine or placebo were given to children in four previously treatment-naïve Liberian villages, 1976-78. Plasmodium falciparum in vivo resistance developed to pyrimethamine only. Selection of molecular markers of P. falciparum resistance after 2 years of treatment are reported. METHODS: Blood samples were collected from 191 study children in a survey in 1978. Polymorphisms in pfcrt, pfmdr1, pfdhfr, pfdhps, pfmrp1 and pfnhe1 genes were determined using PCR-based methods. RESULTS: Pfcrt 72-76 CVIET was found in one chloroquine village sample, all remaining samples had pfcrt CVMNK. Pfmdr1 N86 prevalence was 100%. A pfmdr1 T1069ACT→ACG synonymous polymorphism was found in 30% of chloroquine village samples and 3% of other samples (P = 0.008). Variations in pfnhe1 block I were found in all except the chloroquine treated village (P < 0.001). Resistance associated pfdhfr 108N prevalence was 2% in the pyrimethamine village compared to 45-65% elsewhere, including the placebo village (P = 0.001). CONCLUSIONS: Chloroquine treatment possibly resulted in the development of pfcrt 72-76 CVIET. Selection of pfmdr1 T1069ACG and a pfnhe1 block 1 genotypes indicates that chloroquine treatment exerted a selective pressure on P. falciparum. Pyrimethamine resistance associated pfdhfr 108N was present prior to the introduction of any drug. Decreased pfdhfr 108N frequency concurrent with development of pyrimethamine resistance suggests a non-pfdhfr polymorphisms mediated resistance mechanism

Intercontinental spread of pyrimethamine-resistant malaria.

Here we present molecular evidence demonstrating that malaria parasites bearing high-level pyrimethamine resistance originally arrived in Africa from southeast Asia. The resistance alleles carried by these migrants are now spreading across Africa at an alarming rate, signaling the end of affordable malaria treatment and presenting sub-Saharan Africa with a public health crisis

The genetic change in P. falciparum populations of rural Tanzania resulting from national policy on firstline malaria treatment and pilot Sulfadoxine/pyrimethamine and Artesunate combination

Malaria Journal 2010, 9(Suppl 2):P20Theory predicts that we can protect the efficacy of future antimalarials by changing treatment practice or drug formulation, but the potential success of such interventions rests upon their impact on drug pressure in the field. So far, gathering field data on the relationship between policy, drug pressure, recombination and the evolution of resistance has been entirely challenging. To test these predictions, dhfr and dhps frequency changes were measured in two rural districts of Rufiji and Kilombero/Ulanga during 2000-2006, and the frequencies of the two genes compared prior, during and after antimalarial policy change from first line CQ to first line SP in 2001. Furthermore, while SP first line was maintained in Kilombero/Ulanga, pilot combination therapy of SP+Artesunate (ART) was introduced in Rufiji in 2002 to replace SP and dhfr and dhps frequency changes compared between the two districts. Size polymorphisms at three sets of microsatellite loci linked to dhfr and three other sets of unlinked microsatellite loci were analysed. Genetic analysis of SP resistance genes was carried out on 9,662 Plasmodium falciparum infections identified in a series of annual cross sectional surveys conducted in the two districts between 2000-2006. The frequency of dhfr and dhps resistance alleles did not change significantly while SP was the recommended second-line treatment, but highly significant changes occurred during the subsequent year after the switch to first line SP. The frequency of the triple mutant dhfr allele increased by 37% -63% and that of double mutant dhps allele increased 200%-300%. A strong association between these unlinked alleles also emerged; confirming that they are co-selected by SP. Distribution of major lineages indicates that there is extensive genetic exchange among the geographic regions. Combination therapy had visible effect on the frequencies of dhfr and dhps resistance alleles. The findings of this study provide insight on the interplay between policy, drug pressure, recombination and the evolution of resistance

Differential effect of regional drug pressure on dihydrofolate reductase and dihydropteroate synthetase mutations in southern Mozambique.

The prevalence and frequency of the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) mutations associated with sulfadoxine-pyrimethamine (SP) resistance at 13 sentinel surveillance sites in southern Mozambique were examined regularly between 1999 and 2004. Frequency of the dhfr triple mutation increased from 0.26 in 1999 to 0.96 in 2003, remaining high in 2004. The dhps double mutation frequency peaked in 2001 (0.22) but declined to baseline levels (0.07) by 2004. Similarly, parasites with both dhfr triple and dhps double mutations had increased in 2001 (0.18) but decreased by 2004 (0.05). The peaking of SP resistance markers in 2001 coincided with a SP-resistant malaria epidemic in neighboring KwaZulu-Natal, South Africa. The decline in dhps (but not dhfr) mutations corresponded with replacement of SP with artemether-lumefantrine as malaria treatment policy in KwaZulu-Natal. Our results show that drug pressure can exert its influence at a regional level rather than merely at a national level

Quantification of markers of antimalarial drug resistance from an area of high malaria transmission: Comparing frequency with prevalence

Molecular monitoring of markers of antimalarial drug resistance offers an affordable alternative to the in vivo method for the detection of resistance, and has the potential to guide public health policy in a timely manner. However, the optimal way of analyzing and reporting these data, particularly those emanating from areas of moderate to high malaria transmission, has never been fully explored or agreed upon, given the potential of being confounded by coinfections. By using large number of real field samples, we quantified the difference between prevalence and frequency when reporting field data on antimalarial drug resistance obtained by direct counting of haplotypes. Polymerase chain reaction (PCR) and sequence specific oligonucleotide probing was used to generate point mutations which were used to construct haplotypes. Results indicate that frequency underestimates haplotypes present at low levels while also amplifying haplotypes present at high levels; prevalence on the other hand behaved in a vice versa manner. Both prevalence and frequency are therefore essential, as each may have relevance in different contexts in high malaria transmission settings. Frequency is essential to gauge the impact of intervention on antimalarial drug resistance while prevalence may be more relevant when the aim is to determine parasite clearance. Key words: Molecular markers, polymerase chain reaction (PCR) - sequence specific oligonucleotide probing (SSOP), prevalence, frequency

Media, Health Workers, and Policy Makers' Relationship and Their Impact on Antimalarial Policy Adoption: A Population Genetics Perspective

Drug resistance negatively impacts malaria treatments, making treatment policy revision unavoidable. So far, studies relating sociopolitical and technical issues on policy change with malaria parasite genetic change are lacking. We have quantified the effect of malaria treatment policy on drug pressure and the influence of the media, policy makers, and health worker relationship on parasite population genetic change in Kilombro/Ulanga district. Cross-sectional surveys of asymptomatic infections conducted before, during and after the switch from chloroquine to sulphadoxine/pyrimethamine were used for genetic analysis of SP resistance genes in 4,513 asymptomatic infections identified, and their frequency change was compared with retrospective study of the documented process of policy change. Highly significant changes of dhfr and dhps resistance alleles occurred within one year of switch to SP first line, followed by a decline of their rate of selection caused by reduction of SP usage, as a result of negative media reports on SP usage and lack of adequate preparations

Genetic diversity of next generation antimalarial targets: A baseline for drug resistance surveillance programmes.

Drug resistance is a recurrent problem in the fight against malaria. Genetic and epidemiological surveillance of antimalarial resistant parasite alleles is crucial to guide drug therapies and clinical management. New antimalarial compounds are currently at various stages of clinical trials and regulatory evaluation. Using ?2000 Plasmodium falciparum genome sequences, we investigated the genetic diversity of eleven gene-targets of promising antimalarial compounds and assessed their potential efficiency across malaria endemic regions. We determined if the loci are under selection prior to the introduction of new drugs and established a baseline of genetic variance, including potential resistant alleles, for future surveillance programmes

Surveillance of artemether-lumefantrine associated Plasmodium falciparum multidrug resistance protein-1 gene polymorphisms in Tanzania.

BACKGROUND: Resistance to anti-malarials is a major public health problem worldwide. After deployment of artemisinin-based combination therapy (ACT) there have been reports of reduced sensitivity to ACT by malaria parasites in South-East Asia. In Tanzania, artemether-lumefantrine (ALu) is the recommended first-line drug in treatment of uncomplicated malaria. This study surveyed the distribution of the Plasmodium falciparum multidrug resistance protein-1 single nucleotide polymorphisms (SNPs) associated with increased parasite tolerance to ALu, in Tanzania. METHODS: A total of 687 Plasmodium falciparum positive dried blood spots on filter paper and rapid diagnostic test strips collected by finger pricks from patients attending health facilities in six regions of Tanzania mainland between June 2010 and August 2011 were used. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was used to detect Pfmdr1 SNPs N86Y, Y184F and D1246Y. RESULTS: There were variations in the distribution of Pfmdr1 polymorphisms among regions. Tanga region had exceptionally high prevalence of mutant alleles, while Mbeya had the highest prevalence of wild type alleles. The haplotype YFY was exclusively most prevalent in Tanga (29.6%) whereas the NYD haplotype was the most prevalent in all other regions. Excluding Tanga and Mbeya, four, most common Pfmdr1 haplotypes did not vary between the remaining four regions (χ² = 2.3, p = 0.512). The NFD haplotype was the second most prevalent haplotype in all regions, ranging from 17% - 26%. CONCLUSION: This is the first country-wide survey on Pfmdr1 mutations associated with ACT resistance. Distribution of individual Pfmdr1 mutations at codons 86, 184 and 1246 varies throughout Tanzanian regions. There is a general homogeneity in distribution of common Pfmdr1 haplotypes reflecting strict implementation of ALu policy in Tanzania with overall prevalence of NFD haplotype ranging from 17 to 26% among other haplotypes. With continuation of ALu as first-line drug this haplotype is expected to keep rising, thus there is need for continued pharmacovigilance studies to monitor any delayed parasite clearance by the drug