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

Clinical trials.

Plasmodium falciparum DNA was available from two clinical trials in Kampala. The first was a longitudinal comparison of the efficacies of three combination regimens against uncomplicated malaria in children aged 1 to 10 years at enrollment (601 enrolled from November 2004 to June 2005 and an additional 89 enrolled from January to May 2007) (11, 17). In this trial, patients were offered all medical care through the study clinic, so use of antimalarial drugs outside the study protocol was uncommon. Children were treated with quinine only for complicated malaria or after failure of a combination regimen, both uncommon events (17). Samples were collected for parasite culture between August 2006 and May 2008. The second trial was a randomized comparison of quinine and artemether-lumefantrine for the treatment of uncomplicated falciparum malaria in children aged 6 to 59 months (1); 86 children were enrolled into the quinine treatment arm of the study. Responses over 28 days were graded on the basis of standard WHO criteria, with genotyping used to distinguish recurrences due to reinfection from those due to recrudescence (1). In both trials, when falciparum malaria was diagnosed on the basis of fever or a history of fever and parasites on Giemsa-stained blood smears, blood spots were collected on filter paper (Whatman 3MM) for subsequent molecular studies. All study subjects provided written informed consent. Both trials and the analysis of cultured parasites were approved by the Uganda National Council for Science and Technology, the Makerere University Research and Ethics Committee, and the University of California, San Francisco, Committee on Human Research.

Parasite culture and measurement of in vitro quinine sensitivity.

For the first trial described above, at the time of diagnosis of an episode of malaria and before the initiation of therapy, blood was collected in heparinized tubes and transported within 30 min to our laboratory. Giemsa-stained thin blood smears were examined, and if P. falciparum monoinfection was confirmed, culture was initiated. Blood was centrifuged, plasma and buffy coat were removed, and the erythrocyte pellet was washed twice with RPMI 1640 medium at 37°C. Parasites were diluted with 2% group O uninfected erythrocytes to obtain a density of 0.05%. Aliquots (200 μl) were then cultured in 10 ml RPMI 1640 medium supplemented with 25 mM HEPES, 0.2% NaHCO₃, 0.1 mM hypoxanthine, 100 μg/ml gentamicin, and 0.5% Albumax II serum substitute to produce a packed cell volume of ∼2%. Sensitivities were measured for multiple drugs, including quinine, as previously described utilizing 96-well culture plates predosed with serial dilutions of drugs and a histidine-rich protein 2 (HRP-2)-based enzyme-linked immunosorbent assay (ELISA) (29). For this assay, optical density values were fitted to normal curves based on serial dilutions of HRP-2 standards, and IC₅₀s were calculated on the basis of a nonlinear regression model.

Analysis of parasite genetic polymorphisms.

DNA was extracted from filter paper samples by extraction with Chelex, as previously described (35). We screened for polymorphisms at pfcrt K76T and pfmdr1 N86Y, Y184F, S1034C, N1042D, and D1246Y by PCR amplification of flanking sequences, sequence-specific restriction enzyme digestion, and evaluation of the digested fragments by agarose gel electrophoresis, all as previously reported (12, 16). Estimates of pfmdr1 gene copy number were carried out by quantitative PCR as previously reported (14, 39), using a 7500 real-time PCR system (Applied Biosystems).

The ms4760 locus of the pfnhe1 gene was amplified with primers NHE-A (5′-AGTCGAAGGCGAATCAGATG-3′) and NHE-B (5′-GATACTTACGAACATGTTCATG-3′) (53), using a Titanium PCR kit (Clontech). Each 20-μl PCR mixture included 14.8 μl H₂O, 2 μl 10× buffer, 0.4 μl deoxynucleoside triphosphate mix (10 mM each), 0.2 μl of each 20 μM primer, 0.4 μl 50× Titanium Taq, and 2 μl of target DNA template. A touchdown PCR approach (23) used the following parameters: 94°C for 120 s; 10 cycles of 94°C for 15 s, 63°C for 30 s, and 68°C for 90 s; 25 cycles of 94°C for 15 s, 58°C for 30 s, and 68°C for 90 s; and 1 cycle of 68°C for 7 min. The PCRs were performed on a Bio-Rad C1000 or S1000 thermocycler. PCR products were purified using ExoSAP-IT (USB). About 150 ng of each purified DNA was sequenced using 15 pmol primers NHE-C (5′-ATCCCTGTTGATATATCGAATG-3′) and NHE-D (5′-TTGTCATTAGTACCCTTAGTTG-3′) (53) at the University of California, San Francisco, Genomics Core Facility using ABI BigDye (version 3.1) Terminator sequencing chemistry and an ABI Prism 3730xl capillary DNA analyzer (Applied Biosystems).

For analysis of pfnhe1 ms4760, nucleotide traces were physically inspected using the SeqMan tool (DNAStar Lasergene 8 software). The sequences were then translated using the Edit Sequence tool and aligned using the MEGALIGN program (DNAStar). Microsatellite 4760 profiles that were not among the 35 previously reported profiles (5, 18, 25, 53), were confirmed by repeat template amplification and sequencing.

Statistical analysis.

Quinine IC₅₀s were calculated using a polynomial regression model and HN-NonLin software (http://malaria.farch.net) and were considered continuous variables. Differences in quinine IC₅₀s were examined using the Mann-Whitney U test for comparisons of two groups or the Kruskal-Wallis test with Dunn's multiple-comparison posttest for comparisons of more than two groups. Infections with mixed mutant/wild-type genotypes at the pfcrt and pfmdr1 loci were analyzed as mutant, in view of the expected phenotype of the infection. Associations between alleles and treatment outcomes were assessed using Fisher's exact two-tailed test. Tests were carried out using GraphPad Prism software. In all cases the statistical significance level was set at a P value of <0.05.

Nucleotide sequence accession numbers.

Nucleotide sequences for the Ugandan isolates were deposited in the GenBank database under accession numbers HQ412347 to HQ412386.

Samples available for study.

We evaluated polymorphisms in two sets of samples from patients infected with P. falciparum in Kampala. First, we evaluated samples from a cohort of children who were followed in a drug efficacy trial comparing three combination regimens for uncomplicated falciparum malaria (17). Blood samples collected upon presentation with uncomplicated falciparum malaria and before the initiation of therapy were used to inoculate cultures, and in vitro sensitivities to quinine were determined immediately after sample collection (29). Second, we evaluated 79 samples from the quinine treatment arm of a trial with children randomized to treatment of uncomplicated falciparum malaria with either artemether-lumefantrine or quinine (1). Patients were followed over 28 days after presentation, and treatment outcomes were classified on the basis of WHO criteria (54); in vitro sensitivities were not assessed for samples from this trial.

Associations between polymorphisms and in vitro quinine sensitivity.

The in vitro sensitivities to quinine of isolates collected in Kampala varied widely, with a geometric mean IC₅₀ of 94.4 nM and an IC₅₀ range of from 15 to 761 nM (29). We first assessed known polymorphisms in pfcrt and pfmdr1 and searched for associations with the sensitivity of cultured parasites to quinine. The pfcrt 76T mutation was seen in all except 1 of 169 tested samples. Thus, many parasites were highly sensitive to quinine, despite the presence of pfcrt 76T, and it was not possible to assess the role of this polymorphism in quinine sensitivity. For pfmdr1, the N86Y and 1246Y mutations were also very common, although they were not fixed; the 184F mutation was less common; and mutations at positions 1034 and 1042, which have principally been described in samples from regions other than Africa, were not seen (Table 1). For the 86Y, 184F, and 1246Y polymorphisms, mutant parasites had diminished sensitivity to quinine (Table 1), but none of these differences attained statistical significance. Considering combinations of polymorphisms, there was a trend toward decreased quinine sensitivity with increased numbers of mutations (Table 2). The difference was statistically significant, after adjustment for multiple comparisons, between isolates having any 1 of the pfmdr1 mutations and isolates having all 3 pfmdr1 mutations (P = 0.02). Increased copy number of pfmdr1 has been associated with diminished quinine sensitivity in Asia but has been seen uncommonly in African isolates. To consider a potential contribution of pfmdr1 copy number on results from Uganda, we analyzed 58 samples with a range of quinine sensitivities (26 samples with IC₅₀s of from 15 to 22 nM, 10 with IC₅₀s of from 41 to 298 nM, 22 with IC₅₀s of from 308 to 755 nM). All of these parasites contained only a single copy of the pfmdr1 gene.

We next evaluated associations between polymorphisms in pfnhe1 ms4760 and quinine sensitivity. We sequenced 240 isolates, and in vitro quinine sensitivity data were obtained for 172 isolates. We found a high degree of polymorphism at this locus. We found 46% (16/35) of the previously described ms4760 haplotypes. In addition, we identified 24 previously undescribed ms4760 haplotypes, now designated ms4760-36 to ms4760-59 (Table 3 and Fig. 1). Nucleotide sequences for the Ugandan isolates were deposited in the GenBank database under the accession numbers indicated above and in Fig. 1. The number of DNNND repeats ranged from 1 to 6 (mean ± standard deviation [SD], 2.12 ± 0.86), and that of DDNHNDNHNND repeats ranged from 1 to 3 (mean ± SD, 1.83 ± 0.54). The quinine sensitivities of isolates with different numbers of DNNND and DDNHNDHNND repeats were compared. Sensitivities varied greatly within each category, but parasites with 2 copies of either repeat were somewhat less sensitive to quinine than those with 1 or 3 or more repeats (Table 4), although the associations between repeat number and quinine IC₅₀ were not significant (P = 0.20 and 0.15 for DNNND and DDNHNDHNND, respectively). Considered together, the impacts of known polymorphisms in pfcrt, pfmdr1, and pfnhe1 did not lead to identification of significant associations. Considering the combinations leading to the least and the most sensitive parasites, those with the wild-type sequence at pfmdr1 positions 86 and 1246 and 3 DNNND repeats were more sensitive to quinine (IC₅₀, 55 nM) than parasites with mutant pfmdr1 sequences and 2 DNNND repeats (IC₅₀, 151 nM), but the differences were not statistically significant (P = 0.13; Table 5).

Associations between polymorphisms and quinine treatment responses.

Of 79 samples available from the quinine treatment arm of a recent drug effectiveness trial in Kampala (1), we excluded 12 samples from patients classified by genotyping as clinical failures due to new infection during follow-up and 1 due to failed amplification, leaving 66 samples for analysis, including 46 (70%) from patients who experienced adequate clinical and parasitological responses and 20 (30%) from treatment failures due to recrudescent parasites (2 early treatment failures, 11 late clinical failures, and 7 late parasitological failures). All evaluable isolates (2 failed to amplify) had the pfcrt 76T mutation. For pfmdr1, the prevalence rates of the 86Y, 184F, and 1246Y mutations were very similar between isolates that led to adequate response or treatment failure (Fig. 2). Combinations of any 2 or 3 pfmdr1 mutations were also not associated with treatment response. As with isolates collected for in vitro analyses, mutations at codons 1034 and 1042 (tested in all 66 isolates) or increases in copy number (tested in 55 samples) of pfmdr1 were not seen in any of the clinical isolates.

We next considered the impact of polymorphisms in pfnhe1 on treatment responses. The numbers of DNNND and DDNHNDHNND repeats varied somewhat between groups, but there was no significant association between the number of repeats and clinical response (Fig. 3). In particular, there was no significant difference in quinine treatment response between individuals harboring parasites with one versus two or more DNNND or DDNHNDNHNND repeats.