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.

BACKGROUND: Malaria remains a major public health problem, and its control has been hampered by drug resistance. For a number of drugs, Plasmodium falciparum single nucleotide polymorphisms (SNPs) are associated with altered drug sensitivity and can be used as markers of drug resistance. Several techniques have been studied to assess resistance markers. The most widely used methodology is restriction fragment length polymorphism (RFLP) analysis. The ligase detection reaction fluorescent microsphere (LDR-FM) assay was recently shown to provide high throughput assessment of P. falciparum SNPs associated with drug resistance. The aim of this study was to validate the reliability and accuracy of the LDR-FM assay in a field setting. METHODS: For 223 samples from a clinical trial in Tororo, Uganda in which P. falciparum was identified by blood smear, DNA was extracted from dried blood spots, genes of interest were amplified by PCR, amplicons were analysed by both RFLP and LDR-FM assays, and results were compared. RESULTS: SNP prevalence (wild type/mixed/mutant) with RFLP analysis was 8/5/87% for pfcrt K76T, 34/37/29% for pfmdr1 N86Y, 64/17/19% for pfmdr1 Y184F, and 42/21/37% for pfmdr1 D1246Y. These prevalences with the LDR-FM assay were 7/5/88%, 31/24/45%, 62/20/18%, and 48/19/33% for the four SNPs, respectively. Combining mixed and mutant outcomes for analysis, agreement between the assays was 97% (K=0.77) for pfcrt K76T, 79% (K=0.55) for pfmdr1 N86Y, 83% (K=0.65) for pfmdr1 Y184F, and 91% (K=0.82) for pfmdr1 D1246Y, with most disagreements due to discrepant readings of mixed genotypes. CONCLUSION: The LDR-FM assay provides a high throughput, relatively inexpensive and accurate assay for the surveillance of P. falciparum SNPs associated with drug resistance in resource-limited countries.

A number of human genetic polymorphisms are prevalent in tropical populations and appear to offer protection against symptomatic and/or severe malaria. We compared the prevalence of four polymorphisms, the sickle hemoglobin mutation (β globin E6V), the α-thalassemia 3.7kb deletion, glucose-6-phosphate dehydrogenase deficiency caused by the common African variant (G6PD A-), and the CD36 T188G mutation in 1344 individuals residing in districts in eastern (Tororo), south-central (Jinja), and southwestern (Kanungu) Uganda. Genes of interest were amplified, amplicons subjected to mutation-specific restriction endonuclease digestion (for sickle hemoglobin, G6PD A-, and CD36 T188G), reaction products resolved by electrophoresis, and genotypes determined based on the sizes of reaction products. Mutant genotypes were common, with many more heterozygous than homozygous alleles identified. The prevalences (heterozygotes plus homozygotes) of sickle hemoglobin (28% Tororo, 25% Jinja, 7% Kanungu), α-thalassemia (53% Tororo, 45% Jinja, 18% Kanungu) and G6PD A- (29% Tororo, 18% Jinja, 8% Kanungu) were significantly greater in Tororo and Jinja compared to Kanungu (p<0.0001 for all three alleles); prevalences were also significantly greater in Tororo compared to Jinja for α-thalassemia (p=0.03) and G6PD A- (p<0.0001). For the CD36 T188G mutation, the prevalence was significantly greater in Tororo compared to Jinja or Kanungu (27% Tororo, 17% Jinja, 18% Kanungu; p=0.0004 and 0.0017, respectively). Considering ethnicity of study subjects, based on primary language spoken, the prevalence of mutant genotypes was lower in Bantu compared to non-Bantu language speakers, but in the Jinja cohort, the only study population with a marked diversity of language groups, prevalence did not differ between Bantu and non-Bantu speakers. These results indicate marked differences in human genetic features between populations in different regions of Uganda. These differences might be explained by both ethnic variation and by varied malaria risk in different regions of Uganda.

We measured susceptibilities of Ugandan Plasmodium falciparum isolates assayed on the day of collection or after storage at 4°C. Samples were incubated with serial dilutions of 8 antimalarials, and susceptibilities were determined from 72-h growth inhibition assays. Storage was associated with decreased growth and lower 50% inhibitory concentration values, but differences between assays beginning on day 0 or after 1 or 2 days of storage were modest, indicating that short-term storage before drug susceptibility determination is feasible.

Background

Plasmodium falciparum genetic polymorphisms that mediate altered drug sensitivity may impact upon virulence. In a cross-sectional study, Ugandan children with infections mutant at pfcrt K76T, pfmdr1 N86Y, or pfmdr1 D1246Y had about one-fourth the odds of symptomatic malaria compared to those with infections with wild type (WT) sequences. However, results may have been confounded by greater likelihood in those with symptomatic disease of higher density mixed infections and/or recent prior treatment that selected for WT alleles.

Methods

Polymorphisms in samples from paired episodes of asymptomatic and symptomatic parasitaemia in 114 subjects aged 4-11 years were followed longitudinally in Tororo District, Uganda. Paired episodes occurred within 3-12 months of each other and had no treatment for malaria in the prior 60 days. The prevalence of WT, mixed, and mutant alleles was determined using multiplex ligase detection reaction-fluorescent microsphere assays.

Results

Considering paired episodes in the same subject, the odds of symptomatic malaria were lower for infections with mutant compared to WT or mixed sequence at N86Y (OR 0.26, 95% CI 0.09-0.79, p = 0.018), but not K76T or D1246Y. However, symptomatic episodes (which had higher densities) were more likely than asymptomatic to be mixed (for N86Y OR 2.0, 95% CI 1.04-4.0, p = 0.036). Excluding mixed infections, the odds of symptomatic malaria were lower for infections with mutant compared to WT sequence at N86Y (OR 0.33, 95% CI 0.11-0.98, p = 0.046), but not the other alleles. However, if mixed genotypes were grouped with mutants in this analysis or assuming that mixed infections consisted of 50% WT and 50% mutant genotypes, the odds of symptomatic infection did not differ between infections that were mutant or WT at the studied alleles.

Conclusions

Although infections with only the mutant pfmdr1 86Y genotype were associated with symptomatic infection, this association could primarily be explained by greater parasite densities and therefore greater prevalence of mixed infections in symptomatic children. These results indicate limited association between the tested polymorphisms and risk of symptomatic disease and highlight the value of longitudinal studies for assessing associations between parasite factors and clinical outcomes.

We explored associations between Plasmodium falciparum drug resistance-mediating polymorphisms and clinical presentations in parasitemic children enrolled in a cross-sectional survey in Tororo, Uganda, using a retrospective case-control design. All 243 febrile children (cases) and 243 randomly selected asymptomatic children (controls) were included. In a multivariate analysis adjusting for age, complexity of infection, and parasite density, the prevalence of wild-type genotypes was significantly higher in febrile children compared to asymptomatic children (pfcrt K76T: odds ratio [OR] 4.41 [95% confidence interval {CI}, 1.28-15.1]; pfmdr1 N86Y: OR 4.08 [95% CI, 2.01-8.31], and pfmdr1 D1246Y: OR 4.90 [95% CI, 1.52-15.8]), suggesting greater virulence for wild-type parasites.

We evaluated markers of artemisinin resistance in Plasmodium falciparum isolated in Kampala in 2014. By standard in vitro assays, all isolates were highly sensitive to dihydroartemisinin (DHA). By the ring-stage survival assay, after a 6-h DHA pulse, parasitemia was undetectable in 40 of 43 cultures at 72 h. Two of 53 isolates had nonsynonymous K13-propeller gene polymorphisms but did not have the mutations associated with resistance in Asia. Thus, we did not see evidence for artemisinin resistance in Uganda.

The potential spread of antimalarial drug resistance to Africa, in particular for artemisinins and key partner drugs, is a major concern. We surveyed Plasmodium falciparum genetic markers associated with drug sensitivity on 3 occasions at ∼6-month intervals in 2016 and 2017 at 10 sites representing a range of epidemiological settings in Uganda. For putative drug transporters, we found continued evolution toward wild-type sequences associated with increased sensitivity to chloroquine. For pfcrt K76T, by 2017 the prevalence of the wild type was >60% at all sites and >90% at 6 sites. For the pfmdr1 N86Y and D1246Y alleles, wild type prevalence ranged from 80 to 100%. We found low prevalence of K13 propeller domain mutations, which are associated with artemisinin resistance in Asia, but one mutation previously identified in northern Uganda, 675V, was seen in 2.0% of samples, including 5.5% of those from the 3 northernmost sites. Amplification of the pfmdr1 and plasmepsin2 genes, associated elsewhere with decreased sensitivity to lumefantrine and piperaquine, respectively, was seen in <1% of samples. For the antifolate targets pfdhfr and pfdhps, 5 mutations previously associated with resistance were very common, and the pfdhfr 164L and pfdhps 581G mutations associated with higher-level resistance were seen at multiple sites, although prevalence did not clearly increase over time. Overall, changes were consistent with the selective pressure of the national treatment regimen, artemether-lumefantrine, with increased sensitivity to chloroquine, and with poor efficacy of antifolates. Strong evidence for resistance to artemisinins was not seen. Continued surveillance of markers that predict antimalarial drug sensitivity is warranted.

Background

Multiple red blood cell (RBC) variants appear to offer protection against the most severe forms of Plasmodium falciparum malaria. Associations between these variants and uncomplicated malaria are less clear.

Methods

Data from a longitudinal cohort study conducted in 3 sub-counties in Uganda was used to quantify associations between three red blood cell variants Hb [AA, AS, S (rs334)], alpha thalassaemia 3.7 kb deletion, and glucose-6-phosphate dehydrogenase deficiency A-(G6PD 202A genotype) and malaria incidence, parasite prevalence, parasite density (a measure of anti-parasite immunity) and body temperature adjusted for parasite density (a measure of anti-disease immunity). All analyses were adjusted for age, average household entomological inoculation rate, and study site. Results for all variants were compared to those for wild type genotypes.

Results

In children, HbAS was associated, compared to wild type, with a lower incidence of malaria (IRR = 0.78, 95% CI 0.66-0.92, p = 0.003), lower parasite density upon infection (PR = 0.66, 95% CI 0.51-0.85, p = 0.001), and lower body temperature for any given parasite density (- 0.13 ℃, 95% CI - 0.21, - 0.05, p = 0.002). In children, HbSS was associated with a lower incidence of malaria (IRR = 0.17, 95% CI 0.04-0.71, p = 0.02) and lower parasite density upon infection (PR = 0.31, 95% CI 0.18-0.54, p < 0.001). α-/αα thalassaemia, was associated with higher parasite prevalence in both children and adults (RR = 1.23, 95% CI 1.06-1.43, p = 0.008 and RR = 1.52, 95% CI 1.04-2.23, p = 0.03, respectively). G6PD deficiency was associated with lower body temperature for any given parasite density only among male hemizygote children (- 0.19 ℃, 95% CI - 0.31, - 0.06, p = 0.003).

Conclusion

RBC variants were associated with non-severe malaria outcomes. Elucidation of the mechanisms by which they confer protection will improve understanding of genetic protection against malaria.

We assessed Plasmodium falciparum drug resistance markers in parasites collected in 2012, 2013, and 2015 at 3 sites in Uganda. The prevalence and frequency of parasites with mutations in putative transporters previously associated with resistance to aminoquinolines, but increased sensitivity to lumefantrine (pfcrt 76T; pfmdr1 86Y and 1246Y), decreased markedly at all sites. Antifolate resistance mutations were common, with apparent emergence of mutations (pfdhfr 164L; pfdhps 581G) associated with high-level resistance. K13 mutations linked to artemisinin resistance were uncommon and did not increase over time. Changing malaria treatment practices have been accompanied by profound changes in markers of resistance.