Rapid detection of Pfcrt and Pfmdr1 mutations in Plasmodium falciparum isolates by FRET and in vivo response to chloroquine among children from Osogbo, Nigeria

While there is an active search for new antimalarial drug combinations that could prevent or delay further spread of resistance, there is a need to understand the basis of parasites resistance to chloroquine (CQ) and other antimalarial drugs and explore potentials to use the data in improving the potency and rational for selecting components for effective drug combination. Constant observation of the existing parasite population concerning their genetic makeup determining the resistance to CQ became even more important since it was shown that after CQ withdrawal for therapy CQ-sensitive parasite re-occurred [1]. So written-off drugs may come into focus again.

The molecular basis of CQ resistance in Plasmodium falciparum is still unclear, and the association of point mutations in different genes with chloroquine-resistance has been largely studied in the last decade. In 2000, pfcrt gene was identified [2]. This gene consisting of 13 exons showed 6–8 point mutations including one that appears to play a crucial role in CQR [3]. A lysine to threonine change at position 76 (K76T) which was subsequently found in every in vitro CQR parasite from around the world [4, 5] was identified as an important mutation associated with CQR. The resistance was associated with a reduced accumulation of CQ in the parasite digestive vacuole but how the pfcrt gene exerts such an effect on the digestive vacuole is still unclear. Many studies have shown that the pfcrt play a crucial on CQR, but this mutation was not the sole requirement, suggesting that other factors including host factors are responsible for the clearance of CQR parasites [6].

Polymorphisms in pfmdr1, a gene located on chromosome 5 which encodes the P. falciparum P-glycoprotein homologue-1 is also thought to modulate CQR. It is a typical member of the ATP-binding cassette transporter superfamily localized in the parasite vacuole, where it may regulate intracellular drug concentrations [7]. Mutations were observed at the amino acids 86, 184, 1034, 1042, and 1246, which were strongly linked to the CQR in laboratory clones obtained from various regions [8]. However, the link between pfmdr1 and CQR still remains unclear and controversial [6, 9]. While some field studies had indicated that there is positive association between CQR and mutation (asparagine to tyrosine change) at position 86 (N86Y) [10, 11], several others had doubts about this association [12, 13]. Currently, pfmdr1 mutations are said to assist the CQR parasites by augmenting the level of resistance. A combination of pfcrt and pfmdr1 polymorphisms is believed to result in higher levels of CQR [4, 7].

In Nigeria, CQ has been used for many years as the first-line treatment for uncomplicated malaria. However, like many other malaria endemic regions the therapeutic efficacy of CQ has decreased considerably. This, therefore, has led to the change in the first line drug for the treatment of malaria to artemisinin-based combination, although, CQ is still widely used in the country. In order to explore the roles of pfcrt and pfmdr1 polymorphisms in CQR, the Fluorescence Resonance Energy Transfer (FRET) method was used to determine these polymorphisms and their in vivo sensitivity to chloroquine in P. falciparum isolates from Osogbo Western Nigeria. The use of a Real Time PCR assay for a rapid, sensitive, and specific detection of these mutations was also assessed.

In this study, a Real Time PCR assay for the detection of pfcrt and pfmdr1 alleles thought to be associated with CQ susceptibility and resistance was described. This Real-Time PCR assay was demonstrated to be rapid, sensitive, and specific for the detection and characterization of P. falciparum genetic marker of CQR. The assay detected mixed alleles infections and clearly discriminated between CQ-susceptible and CQ-resistant isolates. Its speed (up to 72 samples can be assayed in a 2-h experiment) and performance characteristics may eliminate the need for more complicated approaches and make it an attractive strategy that could easily be adapted to large-scale studies of drug resistance.

Point mutations in the pfcrt gene and to a lesser extent, in the pfmdr1 gene are thought to be associated with CQR [2, 6]. The goal of this study was to evaluate the utility of these molecular markers as indicators of chloroquine resistance in isolates of P. falciparum obtained from Osogbo western Nigeria using a hybridization probe method on a Real Time PCR technology platform. The result of this study showed a high prevalence of pfcrt T76 (74%). This observation is consistent with the previous reports from various malaria endemic regions where chloroquine has been widely used. A significant association was also found between the overall in vivo rate of treatment failure and the frequency of mutated pfcrt gene in the population (P = 0.004) as already shown previously in western Nigeria [19] and other malaria endemic areas [18]. 93% of the pre-treatment isolates carried the pfcrt T76 and mixed allele while among the post treatment isolate the prevalence was 98%. The facts that this polymorphism was present in all the recrudescence isolates emphasised again the fact that this mutation is important in CQR.

The point mutation of asparagine to tyrosine at codon 86 of pfmdr1 has been associated with CQR in some studies [8, 9] but not in others [12]. In the present study, both the pfmdr1 Y86 and F184 mutations showed no correlation with resistance to chloroquine. Both the wild type and the mutant alleles for each locus were present in both sensitive and resistance isolates. Previously, an association was established between chloroquine-resistance and alleles of the pfmdr1 gene in laboratory isolates obtained from different parts of the world [8]. Others had considered whether this association existed in parasite isolates obtained directly from the field [9, 20]. Their analysis showed that African isolates predominantly possess polymorphism at two alleles, codon-86 and codon-184, with a positive association, although incomplete, between mutation of codon-86 and CQR. A similar association has been found in several studies [4, 11, 21, 22]. Nevertheless, much confusion has surrounded the association of different pfmdr1 alleles to chloroquine resistance because numerous studies have had contradictory results. Transfection studies as well as carefully controlled molecular epidemiologic studies have shown that there are strong associations between pfmdr1 polymorphisms and antimalarial resistance [7]. However, like many other studies [12] the present findings have failed to find such associations because the presence of both wild-type and mutant-type alleles in our samples were largely independent of their in vivo responses. Also the current belief that the combination of pfcrt and pfmdr1 polymorphisms result in higher levels of CQR [4] was not observed in this study. Although a significant association was observed (p = 0.028), it was not in any way stronger than the one observed with pfcrt alone. Analysis of altered gene expression and other mechanisms that may contribute to a resistant phenotype is needed before the role of pfmdr1 can be excluded.

A recent report from Ibadan Nigeria a neighbouring town to Osogbo had suggested an association and linkage disequilibrium between the pfcrt T76 and pfmdr1 Y86 alleles in chloroquine-resistant isolates [19]. On the contrary this study suggested no possible association between these two polymorphic alleles and in vivo chloroquine resistance and that these molecular markers by themselves may not predict in vivo chloroquine resistance.

In summary, the results of this study give further evidence to the reliability of the 76T pfcrt point mutation as a molecular marker for CQ resistance. Conversely, the role of the Y86 and F184 point mutation of pfmdr1 gene in CQ resistance remains elusive. The analysis of the relevant mutations by RT-PCR provides a rapid and reliable method for epidemiological and clinical studies suited for higher throughput.