Materials and Methods

Study siteThis study used samples from a clinical assessment of chloroquine resistance performed in Kampala, Uganda, from August 1998 through March 1999. Kampala is an urban center where malaria is highly endemic, occurring perennially with peaks during the 2 rainy seasons (Ugandan Ministry of Health, unpublished data). Chloroquine is currently the recommended first-line agent for uncomplicated malaria in Uganda

PatientsFull details of the clinical study that provided the samples evaluated in this report have been described elsewhere [9]. Consecutive patients ⩾6 months old with uncomplicated falciparum malaria at a parasite density ⩾2000/μL were enrolled. Patients were treated with standard doses of chloroquine (25 mg/kg base over 3 days) under supervision and were followed up for 14 days. Patient outcomes were classified according to a modified version of the World Health Organization (WHO) 14-day clinical classification system (early treatment failure, late treatment failure, or adequate clinical response) and the WHO parasitologic classification system (sensitive; and RI, RII, and RIII, resistance levels I, II, and III, respectively) [9]

DNA extractionVenous blood was obtained from all patients before treatment and was blotted in ∼50-μL aliquots onto filter paper (no. 3 Whatman), which was stored in sealed plastic bags at room temperature. DNA was extracted with Chelex-100 Resin (Bio-Rad Laboratories), as described elsewhere [10]. For control P. falciparum DNA, genomic DNA was isolated from schizont-stage parasites (cultured by standard methods) by phenol extraction and isopropanol precipitation [11]

Nested polymerase chain reaction (PCR) for the detection ofpfcrt and pfmdr-1 mutationsDetails of PCR reaction conditions are summarized in table 1. Nested PCR protocols were used. The first-round PCR reaction contained 1 μL (∼100 ng) of extracted parasite DNA or 50 ng of control DNA and 1 μM of each primer in 50 μL of 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl₂, and 200 μM dNTP with 2.5 U Taq polymerase (Life Technologies Gibco BRL). For each series of samples, water was used as a negative control, HB3-strain DNA was used as the wild-type control, and Dd2 DNA was used as the mutant control

View larger version:

• In this window
• In a new window

• Download as PowerPoint Slide

Table 1

Polymerase chain reaction protocols for detection of pfcrt and pfmdr-1 point mutations

Two nested PCR methods were used for the detection of point mutations: mutation-specific PCR (MS-PCR) and PCR followed by mutation-specific restriction-enzyme digestion (MS-RED). Both techniques were used for the detection of the pfcrt K76T mutation, MS-PCR was used for the detection of the remaining 7 pfcrt mutations, and MS-RED was used for detection of the 2 pfmdr-1 mutations

Nested 50-μL MS-PCR reactions contained 1:50–1:50,000 dilutions of the first-round amplicon and 1 μM common and mutant-specific or wild-type–specific primers in 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl₂, 200 μM dNTP, and 1.25–2.5 U Taq polymerase. The amplified DNA fragments were resolved by electrophoresis in a 1%–2% agarose gel. Wild-type and mutant genotypes were designated on the basis of amplification of products of predicted sizes by the appropriate primers. If bands were present by means of both wild-type and mutant primers, nested PCR reactions using serial 10-fold dilutions of amplicons from the first-round PCR were performed until only 1 band remained. If both bands disappeared after a single dilution step, the genotype was designated as mixed

For MS-RED, 50-μL reactions contained 1:5000 dilutions of the first-round amplicon and 1 μM each primer in 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl₂, 200 μM dNTP, and 1.25 U Taq polymerase. Aliquots of 5–10 μL of the PCR reaction were incubated with 1–10 U of the restriction enzyme in 100 mM NaCl, 50 mM Tris-HCL, 10 mM MgCl₂, 1 mM dithiothreitol, and 100 μg/μL bovine serum albumin for 6 h at the appropriate temperature. The DNA fragments were resolved by electrophoresis in a 2% agarose gel. For the pfcrt K76T mutation, ApoI cut the wild-type but not mutant gene into 34- and 100-bp fragments. For the pfmdr-1 N86Y mutation, ApoI cut the wild-type gene into 245- and 188-bp fragments, and, for the pfmdr-1 D1246Y mutation, EcoRV cut the mutant gene into 210- and 199-bp fragments. For all 3 reactions, mixed genotypes were designated when 3 bands were present after restriction endonuclease digestion

Statistical analysisStatistical associations between point mutations and in vivo outcomes were assessed using the Fisher’s exact test (2-tailed). P<.05 was considered to be significant

Results

A randomly selected subset of 114 samples from our recent evaluation of chloroquine efficacy in 258 children and adults in Kampala [9] was analyzed for polymorphisms in pfcrt at amino acid 76. These samples were obtained before the initiation of chloroquine therapy and were from patients who subsequently demonstrated a wide range of clinical responses (clinical response was adequate in 56%, treatment failed early in 23%, and treatment failed late in 21%) and parasitologic responses (36% sensitive, 15% RI, 33% RII, and 16% RIII) to the treatment. There was no association (P>.2) between selection of samples for molecular analysis and previously identified predictors of chloroquine resistance (age <5 years, initial temperature ⩾38°C, or chloroquine use 3–14 days before enrollment). All 114 samples, which were analyzed by using both the MS-PCR and MS-RED methods, contained the pfcrt K76T mutation. In no case was a mixed population of parasites identified. Control HB3-strain parasites demonstrated the wild-type sequence with both the MS-PCR and MS-RED methods. Thus, the wild-type K76 sequence was not found in any parasite DNA from Kampala

A subset of 30 samples was selected for a case-control study, to assess associations between chloroquine resistance and known mutations in pfcrt and pfdmr-1. Case-control samples consisted of 15 patients from each extreme of the parasitologic outcome spectrum (i.e., sensitive and RIII) and included only patients <5 years old, to minimize the effect of acquired immunity. For 6 of the 8 studied pfcrt mutations (M74I, N75E, K76T, A220S, Q271E, and R371I), all of the samples with successful amplification revealed only the presence of the mutant sequence (table 2). At 1 site (amino acid 356), only the wild-type sequence was identified. Polymorphisms were identified at only 1 position (amino acid 326). However, at this site, there was no significant association between the sequence and in vivo outcome. Analysis of control wild-type (HB3) and mutant (Dd2) parasite strains yielded the expected sequences in all cases

View larger version:

• In this window
• In a new window

• Download as PowerPoint Slide

Table 2

Correlation of chloroquine resistance outcomes with Plasmodium falciparum genotypes

Investigation of 2 known mutations in the pfmdr-1 gene revealed polymorphisms and a number of mixed genotypes (table 2). However, no sequence at either position was associated significantly with either a sensitive or a resistant clinical outcome

Discussion

In this study, we report on the correlation between polymorphisms in the pfcrt and pfmdr-1 genes of P. falciparum field isolates and in vivo chloroquine response. Our samples were obtained from an area of intense transmission, where chloroquine resistance rates are high [9, 12]. In 114 samples from patients with a wide range of clinical and parasitologic outcomes after therapy with chloroquine, the pfcrt K76T mutation was ubiquitous. All parasite isolates, which were obtained before chloroquine therapy, contained the K76T mutation. A case-control analysis that evaluated 8 known pfcrt mutations and 2 key pfmdr-1 mutations failed to identify any sequence that predicted a sensitive or resistant response to chloroquine therapy in Kampala

Our results suggest that, although the presence of the pfcrt K76T mutation (and perhaps others) may be necessary for the expression of chloroquine resistance, other factors, including host immunity, may play a significant role in determining clinical outcomes after administration of chloroquine. Other recent studies also have shown that many patients with apparently sensitive responses to chloroquine therapy were infected with mutant parasites. At a number of sites in Mali, the prevalence of the K76T mutation was consistently 2–3 times higher than the prevalence of clinical chloroquine resistance (A. Djimde and C. V. Plowe, University of Maryland School of Medicine, Baltimore, personal communication). In Kampala, where the prevalence of clinical chloroquine resistance is higher than in Mali, a similar ratio of genotypic resistance to clinical resistance would predict the presence of the K76T mutation in all parasites, as was seen in our study. Our findings are probably explained by the influence of host immunity on clinical outcomes. In areas of intense transmission, where immunity is high, some patients appear to be able to clear their parasitemia even in the presence of the pfcrt K76T mutation. It has been argued that the emergence of chloroquine resistance only after many years of widespread chloroquine use suggests that multiple mutations are required to produce the chloroquine resistance phenotype [13]. Such mutations might be the multiple identified pfcrt mutations, which may improve the fitness and stability of parasites containing the K76T mutation. Alternatively, our findings are consistent with the possibility that mutations in plasmodial genes other than pfcrt and pfmdr-1 may be important in the development of the chloroquine-resistant phenotype

In summary, our results confirm that PCR-based techniques provide a simple, rapid method of detecting polymorphisms in genes that may affect resistance to chloroquine. However, the predictive value of these tests in areas where levels of clinical chloroquine resistance and transmission are high remains unclear. The development of simple molecular tests to help analyze antimalarial drug resistance in field settings remains an important goal. Therefore, further studies are needed to assess the role of new molecular techniques in the surveillance of antimalarial drug resistance in various epidemiologic settings