Frederick N. Baliraine

Quinine, an old anti-malarial drug in a modern world: role in the treatment of malaria

Quinine remains an important anti-malarial drug almost 400 years after its effectiveness was first documented. However, its continued use is challenged by its poor tolerability, poor compliance with complex dosing regimens, and the availability of more efficacious anti-malarial drugs. This article reviews the historical role of quinine, considers its current usage and provides insight into its appropriate future use in the treatment of malaria. In light of recent research findings intravenous artesunate should be the first-line drug for severe malaria, with quinine as an alternative. The role of rectal quinine as pre-referral treatment for severe malaria has not been fully explored, but it remains a promising intervention. In pregnancy, quinine continues to play a critical role in the management of malaria, especially in the first trimester, and it will remain a mainstay of treatment until safer alternatives become available. For uncomplicated malaria, artemisinin-based combination therapy (ACT) offers a better option than quinine though the difficulty of maintaining a steady supply of ACT in resource-limited settings renders the rapid withdrawal of quinine for uncomplicated malaria cases risky. The best approach would be to identify solutions to ACT stock-outs, maintain quinine in case of ACT stock-outs, and evaluate strategies for improving quinine treatment outcomes by combining it with antibiotics. In HIV and TB infected populations, concerns about potential interactions between quinine and antiretroviral and anti-tuberculosis drugs exist, and these will need further research and pharmacovigilance.

Polymorphisms in Plasmodium falciparum chloroquine resistance transporter and multidrug resistance 1 genes: parasite risk factors that affect treatment outcomes for P. falciparum malaria after artemether-lumefantrine and artesunate-amodiaquine.

Adequate clinical and parasitologic cure by artemisinin combination therapies relies on the artemisinin component and the partner drug. Polymorphisms in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes are associated with decreased sensitivity to amodiaquine and lumefantrine, but effects of these polymorphisms on therapeutic responses to artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) have not been clearly defined. Individual patient data from 31 clinical trials were harmonized and pooled by using standardized methods from the WorldWide Antimalarial Resistance Network. Data for more than 7,000 patients were analyzed to assess relationships between parasite polymorphisms in pfcrt and pfmdr1 and clinically relevant outcomes after treatment with AL or ASAQ. Presence of the pfmdr1 gene N86 (adjusted hazards ratio = 4.74, 95% confidence interval = 2.29 – 9.78, P < 0.001) and increased pfmdr1 copy number (adjusted hazards ratio = 6.52, 95% confidence interval = 2.36–17.97, P < 0.001) were significant independent risk factors for recrudescence in patients treated with AL. AL and ASAQ exerted opposing selective effects on single-nucleotide polymorphisms in pfcrt and pfmdr1. Monitoring selection and responding to emerging signs of drug resistance are critical tools for preserving efficacy of artemisinin combination therapies; determination of the prevalence of at least pfcrt K76T and pfmdr1 N86Y should now be routine.

Prolonged Selection of pfmdr1 Polymorphisms After Treatment of Falciparum Malaria With Artemether-Lumefantrine in Uganda

We compared the prevalence of key pfmdr1 alleles between pretreatment Plasmodium falciparum parasite isolates and parasites that emerged after treatment of uncomplicated malaria in a longitudinal cohort of Ugandan children. The pfmdr1 86N, 184F, and 1246D alleles were selected after treatment with artemether-lumefantrine, but not after artesunate-amodiaquine or amodiaquine-sulfadoxine-pyrimethamine. Remarkably, selection persisted in infections presenting up to about 60 days after treatment with artemether-lumefantrine. Thus, parasites selected for decreased drug sensitivity can appear long after predicted exposure to antimalarial drugs. Continued surveillance of the clinical efficacy and in vitro activity of new combination therapies is warranted.

Temporal Changes in Prevalence of Molecular Markers Mediating Antimalarial Drug Resistance in a High Malaria Transmission Setting in Uganda.

Standard therapy for malaria in Uganda changed from chloroquine to chloroquine + sulfadoxine-pyrimethamine in 2000, and artemether-lumefantrine in 2004, although implementation of each change was slow. Plasmodium falciparum genetic polymorphisms are associated with alterations in drug sensitivity. We followed the prevalence of drug resistance-mediating P. falciparum polymorphisms in 982 samples from Tororo, a region of high transmission intensity, collected from three successive treatment trials conducted during 2003-2012, excluding samples with known recent prior treatment. Considering transporter mutations, prevalence of the mutant pfcrt 76T, pfmdr1 86Y, and pfmdr1 1246Y alleles decreased over time. Considering antifolate mutations, the prevalence of pfdhfr 51I, 59R, and 108N, and pfdhps 437G and 540E were consistently high; pfdhfr 164L and pfdhps 581G were uncommon, but most prevalent during 2008-2010. Our data suggest sequential selective pressures as different treatments were implemented, and they highlight the importance of genetic surveillance as treatment policies change over time.

Genetic structure of Plasmodium vivax and Plasmodium falciparum in the Bannu district of Pakistan.

BackgroundPlasmodium vivax and Plasmodium falciparum are the major causative agents of malaria. While knowledge of the genetic structure of malaria parasites is useful for understanding the evolution of parasite virulence, designing anti-malarial vaccines and assessing the impact of malaria control measures, there is a paucity of information on genetic diversity of these two malaria parasites in Pakistan. This study sought to shed some light on the genetic structure of P. vivax and P. falciparum in this understudied region.MethodsThe genetic diversities of P. vivax and P. falciparum populations from the densely populated, malaria-endemic Bannu district of Pakistan were evaluated by analysis of their merozoite surface protein (msp) genes by PCR-RFLP. Specifically, the Pvmsp-3α and Pvmsp-3β genes of P. vivax and the Pfmsp-1 and Pfmsp-2 genes of P. falciparum were analysed.ResultsIn P. vivax, genotyping of Pvmsp-3α and Pvmsp-3β genes showed a high level of diversity at these loci. Four distinct allele groups: A (1.9 kb), B (1.5 kb), C (1.2 kb), and D (0.3 kb) were detected for Pvmsp-3α, type A being the most prevalent (82%). Conversely, amplification of the P. vivax msp-3β locus produced two allele groups: A (1.7-2.2 kb, 62%) and B (1.4-1.5 kb, 33%), with 5% mixed-strain infections. Restriction analysis of Pvmsp-3α and Pvmsp-3β yielded 12 and 8 distinct alleles, respectively, with a combined mixed genotype prevalence of 20%. In P. falciparum, all three known genotypes of Pfmsp-1 and two of Pfmsp-2 were observed, with MAD20 occurring in 67% and 3D7/IC in 65% of the isolates, respectively. Overall, 24% P. falciparum samples exhibited mixed-strain infections.ConclusionThese results indicate that both P. vivax and P. falciparum populations in Pakistan are highly diverse.

Limited Ability of Plasmodium falciparum pfcrt, pfmdr1, and pfnhe1 Polymorphisms To Predict Quinine In Vitro Sensitivity or Clinical Effectiveness in Uganda

ABSTRACT Quinine is a standard drug for treating severe malaria in Africa, and it is also increasingly used to treat uncomplicated disease. However, failures of quinine therapy are common, and it is unknown if failures in Africa are due to drug resistance. Recent studies have identified associations between in vitro quinine sensitivity and polymorphisms in genes encoding putative transporters, including well-described polymorphisms in pfcrt and pfmdr1 and varied numbers of DNNND or DDNHNDNHNND repeats in microsatellite 4760 (ms4760) of the predicted sodium-hydrogen exchanger, pfnhe1. To better characterize mediators of quinine response, we assessed associations between genetic polymorphisms, in vitro quinine sensitivity, and quinine treatment responses in Kampala, Uganda. Among 172 fresh clinical isolates tested in vitro, decreasing sensitivity to quinine was associated with accumulation of pfmdr1 mutations at codons 86, 184, and 1246. Nearly all parasites had pfcrt 76T, preventing analysis of associations with this mutation. pfnhe1 ms4760 was highly polymorphic. Parasites with 2 copies of either ms4760 repeat showed modest decreases in quinine sensitivity compared to those with 1 or ≥3 repeats, but the differences were not statistically significant. None of the above polymorphisms predicted treatment failure among 66 subjects treated with quinine for uncomplicated malaria. Our data suggest that quinine sensitivity is a complex trait and that known polymorphisms in pfcrt, pfmdr1, and pfnhe1, while associated with quinine sensitivity, are not robust markers for quinine resistance.

Optimization of a Ligase Detection Reaction-Fluorescent Microsphere Assay for Characterization of Resistance-Mediating Polymorphisms in African Samples of Plasmodium falciparum

ABSTRACT Genetic polymorphisms in the malaria parasite Plasmodium falciparum mediate alterations in sensitivity to important antimalarial drugs. Surveillance for these polymorphisms is helpful in assessing the prevalence of drug resistance and designing strategies for malaria control. Multiple methods are available for the assessment of P. falciparum genetic polymorphisms, but they suffer from low throughput, technical limitations, and high cost. We have optimized and tested a multiplex ligase detection reaction-fluorescent microsphere (LDR-FM) assay for the identification of important P. falciparum genetic polymorphisms. For 84 clinical samples from Kampala, Uganda, a region where both transmission intensity and infection complexity are high, DNA was extracted from dried blood spots, genes of interest were amplified, amplicons were subjected to multiplex ligase detection reactions to add bead-specific oligonucleotides and biotin, fragments were hybridized to magnetic beads, and polymorphism prevalences were assessed fluorometrically in a multiplex format. A total of 19 alleles from the pfcrt, pfmdr1, pfmrp1, pfdhfr, and pfdhps genes were analyzed by LDR-FM and restriction fragment length polymorphism (RFLP) analyses. Considering samples with results from the two assays, concordance between the assays was good, with 78 to 100% of results identical at individual alleles, most nonconcordant results differing only between a mixed and pure genotype call, and full disagreement at individual alleles in only 0 to 3% of results. We estimate that the LDR-FM assay offers much higher throughput and lower cost than RFLP. Our results suggest that the LDR-FM system offers an accurate high-throughput means of classifying genetic polymorphisms in field samples of P. falciparum.

A cohort study of Plasmodium falciparum infection dynamics in Western Kenya Highlands

BackgroundThe Kenyan highlands were malaria-free before the 1910s, but a series of malaria epidemics have occurred in the highlands of western Kenya since the 1980s. Longitudinal studies of the genetic structure, complexity, infection dynamics, and duration of naturally acquired Plasmodium falciparum infections are needed to facilitate a comprehensive understanding of malaria epidemiology in the complex Kenyan highland eco-epidemiological systems where malaria recently expanded, as well as the evaluation of control measures.MethodsWe followed a cohort of 246 children residing in 3 villages at altitudes 1430 - 1580 m in western Kenya. Monthly parasitological surveys were undertaken for one year, yielding 866 P. falciparum isolates that were analyzed using 10 microsatellite markers.ResultsInfection complexity and genetic diversity were high (HE = 0.787-0.816), with ≥83% of infections harboring more than one parasite clone. Diversity remained high even during the low malaria transmission season. There was no significant difference between levels of genetic diversity and population structure between high and low transmission seasons. Infection turn-over rate was high, with the average infection duration of single parasite genotypes being 1.11 months, and the longest genotype persistence was 3 months.ConclusionsThese data demonstrate that despite the relatively recent spread of malaria to the highlands, parasite populations seem to have stabilized with no evidence of bottlenecks between seasons, while the ability of residents to clear or control infections indicates presence of effective anti-plasmodial immune mechanisms.

Population structure of Anopheles gambiae along the Kenyan coast.

In the tropics, Anopheles mosquito abundance is greatest during the wet season and decline significantly during the dry season as larval habitats shrink. Population size fluctuations between wet and dry seasons may lead to variation in distribution of specific alleles within natural Anopheles populations, and a possible effect on the population genetic structure. We used 11 microsatellite markers to examine the effect of seasonality on population genetic structure of Anopheles gambiae s.s. at two sites along the Kenyan coast. All loci were highly polymorphic with the total number of alleles for pooled samples ranging from 7 (locus ND36) to 21 (locus AG2H46). Significant estimates of genetic differentiation between sites and seasons were observed suggesting the existence of spatio-temporal subpopulation structuring. Genetic bottleneck analysis showed no indication of excess heterozygosity in any of the populations. These findings suggest that along the Kenyan coast, seasonality and site specific ecological factors can alter the genetic structure of A. gambiae s.s. populations.

Population Genetic Structure of Anopheles Arabiensis (Diptera: Culicidae) in a Rice Growing Area of Central Kenya

ABSTRACT Studies were conducted to examine the population genetic structure of Anopheles arabiensis (Patton) in Mwea Rice Irrigation Scheme and surrounding areas in Central Kenya, under different agricultural systems. This study was motivated by observed differences in malaria transmission indices of An. arabiensis within the scheme compared with adjacent nonirrigated areas. Agricultural practices can modify local microclimate and influence the number and diversity of larval habitats and in so doing may occasion subpopulation differentiation. Thirty samples from each of the three study sites were genotyped at eight microsatellite loci. Seven microsatellite loci showed high polymorphism but revealed no genetic differentiation (FST = 0.006, P = 0.312) and high gene flow (Nm = 29–101) among the three populations. Genetic bottleneck analysis showed no indication of excess heterozygosity in any of the populations. There was high frequency of rare alleles, suggesting that An. arabiensis in the study area has a high potential of responding to selective pressures from environmental changes and vector control efforts. These findings imply that An. arabiensis in the study area occurs as a single, continuous panmictic population with great ability to adapt to human-imposed selective pressures.