Polymorphisms of the pfmdr1 but not the pfnhe-1 gene is associated with in vitro quinine sensitivity in Thai isolates of Plasmodium falciparum

The emergence of anti-malarial resistance in Plasmodium falciparum is a major public health threat worldwide, especially in tropical developing countries. The situation of multidrug-resistant falciparum malaria is most serious along the Thai-Myanmar and Thai-Cambodia borders [1]. To handle this situation, WHO recommends artemisinin derivative-based combination treatment (ACT) for the treatment of uncomplicated falciparum malaria [2]. Artesunate-mefloquine combination has been used as the first-line treatment in Thailand for more than 15 years [3]. Quinine (QN)-tetracycline/doxycycline has been used as the second-line treatment for uncomplicated falciparum malaria in Thailand. In addition, QN monotherapy is the first-line treatment for pregnancy [3, 4]. Unfortunately reduced in vitro and in vivo response to QN has been reported in Southeast Asia [5, 6]. Investigations have been carried out to identify the mechanisms of QN resistance. At least three candidate genes including Plasmodium falciparum chloroquine resistance transporter (pfcrt), Plasmodium falciparum multidrug resistance 1 (pfmdr1), and Plasmodium falciparum Na ⁺ /H ⁺ exchanger (pfnhe-1) have been linked to reduced QN sensitivity [7‐12]. Cooper et al. (2002) showed the association between the pfcrt mutation at codon 76 and QN sensitivity [7]. In addition, recent studies have shown that parasites containing a novel mutation in the pfcrt gene, Q352K/R, C350R, altered QN sensitivity [8, 9]. Concerning the pfmdr1 gene, it has been shown that both mutations and copy number influenced in vitro QN sensitivity [10‐12]. Using quantitative trait loci on the genetic cross of HB3 and Dd2 strains, an additional candidate gene, pfnhe-1, for QN resistance was identified [13]. This gene encodes a 226 kDa parasite plasma membrane protein containing 12 transmembrane domains, and 3 microsatellite regions, msR1, ms3580 and ms4760. Variations in QN susceptibilities between different parasite strains have also been linked to repeat polymorphisms in the microsatellite locus ms4760 of pfnhe-1[13]. A few studies showed that the number of DNNND, DDNNNDNHNDD, and NHNDNHNNDDD repeats in the microsatellite ms4760 influenced in vitro QN sensitivity [14‐19]. However, there is a lack of consensus regarding the specific nature of these associations [20‐25]. For instance, while some studies report an association between reduced susceptibility to QN and an increase in the number of 'DNNND' repeats in ms4760 [15, 18, 19], others could not verify this association [22‐24], or found that two DNNND repeats was the optimal number for conferring a reduction in QN sensitivity [17]. Moreover, amplification of a second ms4760 repeat 'NHNDNHNNDDD' has been linked to increases in the parasite's susceptibility to QN [15, 18, 19]. However, the reverse association has also been reported [22], and several studies have failed to confirm either of these findings [23, 24]. Considering the situation of multidrug-resistant P. falciparum in Thailand, determination of the molecular basis of QN resistance is crucial to be able to monitor parasite resistance. This study was to investigate the influence of the pfmdr1 and pfnhe-1 genes on in vitro QN sensitivity of Thai isolates of P. falciparum from both the Thai-Myanmar and Thai-Cambodia borders.

In the present study, using the IC₅₀ of >500 nM as the cut-off point for in vitro QN resistance, only 4.7% (4/85) exhibited QN resistance. Although a higher cut-off point at 800 nM has been proposed, no parasite isolate was found showing QN IC₅₀ of >800 nM. This result is similar to those previous reports showing that most Thai isolates of P. falciparum were QN sensitive [6, 31, 32]. However data from these studies might not be comparable since different methods including schizont maturation inhibition, isotopic and SYBR green 1 based fluorescence assays were used for the determination of QN IC₅₀. In addition, since culture-adapted isolates were used in some studies including the present study, specific phenotypes might be selected during the adaptation process. The situation of QN resistance in Thailand is less serious than those found in mefloquine and chloroquine. This may be due to a lower drug pressure of QN since it has a shorter half life. However, a decline in QN sensitivity of P. falciparum isolated from the Thai-Myanmar border has been indicated in recent study [6].

All parasite isolates in this study contained chloroquine-resistant haplotype, CVIET of the pfcrt. The association between in vitro QN sensitivity and polymorphisms of the pfmdr1 gene, but not the pfnhe-1 gene, was identified. Although a genetic cross study indicated that QN sensitivity can be modulated by the pfnhe-1 gene [13], the role of the pfnhe-1 gene as a molecular marker for QN resistance is still controversial. Some but not all in vitro and in vivo studies identified the association between DNNND, DDNNNDNHNDD, and NHNDNHNNDDD repeats of the pfnhe-1 ms4760 microsatellite and QN sensitivity and treatment outcome, respectively [14‐19, 23‐25]. Nearly half of Thai isolates in the present study contained 4 DNNND repeats while most parasites from other areas in Southeast Asia including Vietnam and the China-Myanmar border contained 3 DNNND repeats [18, 19]. Similar to these 2 studies, most isolates in the present study contained 1 repeat of NHNDNHNNDDD. The studies with positive association usually showed that parasites with 2 or more than 2 DNNND repeats had a significantly reduced QN sensitivity compared with those with 1 repeat. This association has been found in the studies from Vietnam and the China-Myanmar border as well [18, 19]. In contrast, no significant difference of QN IC₅₀ between parasites containing ≥2 and less DNNND repeats was identified. Inconsistent findings of the association between the response to QN and pfnhe-1 gene might be due to its interaction with other genes such as pfcrt and pfmdr1. This postulation has been provided to explain the result of a knockdown pfnhe-1 expression resulting in increased QN sensitivity in 2 of 3 parasite lines [16]. In addition, a recent study in Kenya found no significant difference of QN IC₅₀ among parasites with different DNNND repeats [17]. However parasites containing 2 DNNND repeats with 86Y pfmdr1 showed a decrease in QN sensitivity. When the QN IC₅₀ of the parasites containing a similar number of DNNND repeats in the pfnhe-1 ms4760 microsatellite with different 86 alleles in the pfmdr1 gene was compared, no significant difference of QN IC₅₀ among these parasites was identified (data not shown). In addition, no significant correlations between the number of DDNNNDNHNDD, and NHNDNHNNDDD repeats of the pfnhe-1 ms4760 microsatellite and in vitro QN sensitivity were found. Since there were only four isolates exhibiting reduced QN susceptibility, this may be restrictive for identification or validation of these new markers.

In contrast to the pfnhe-1 gene, in vitro QN sensitivity was significantly associated with the mutations in the pfmdr1 gene in these Thai isolates. The parasites containing the pfmdr1 184F and 1042D allele showed less sensitivity to QN while those with the pfmdr1 86Y exhibited increased QN sensitivity. However, when the parasites were categorized according to their haplotypes of the pfmdr1 gene, the pfmdr1 86Y and 1042D allele influenced QN sensitivity. Compelling evidence for a significant role of N1042D mutation on in vitro QN sensitivity has been shown in a few studies using allelic exchange strategies [10, 11]. Reed et al. [10] showed that insertion of the pfmdr1 gene containing the 1034C, 1042D and 1246Y alleles made a QN - sensitive parasite become more resistant to QN [10]. Conversely, a QN -resistant line became more sensitive to QN after these alleles were removed. More recently, a study by Sidhu et al. (2005) identified that a single mutation, the N1042D, could modulate the parasites become less sensitive to QN [11]. Unlike the N1042D mutation, the functional role of the N86Y mutation on QN sensitivity has been explored by expression of the pfmdr1 gene in a heterologous system, Xenopus oocytes [33]. Substituting the asparagines (N) at position 86 for tyrosine (Y) resulted in a loss of QN transport ability. Since the site of QN action is in the food vacuole, [34, 35] transport ability of the wild-type Pgh1 reduces drug concentration in the food vacuole, and consequently results in decreased QN susceptibility. This finding is compatible to that found in a few studies using parasites isolated from Southeast Asia, including the present study showing that the parasite isolates containing the pfmdr1 86Y allele showed more sensitive to QN [20, 32]. However, a contrary result was shown in the study of parasite isolates from Kenya [17]. The decrease in QN susceptibility was associated with the pfmdr1 86Y allele in parasites harbouring the two DNNND repeats in the pfnhe-1 ms4760 microsatellite. Since reduced susceptibility to QN appear to be governed by a number of proteins whose contributions vary between strains. The contrasting findings might be explained by different variations in genetic background of parasites from different geographical areas. The influence of the pfmdr1 copy number on QN sensitivity has been confirmed by the study of Sidhu et al. using the knockdown strategy [12]. A few studies of parasite isolates from the Thai-Myanmar border also showed the influence of pfmdr1 copy number on in vitro QN sensitivity. However, this association has not been identified in some studies [21, 22]. In this study, no association between the pfmdr1 copy number and in vitro QN sensitivity neither in parasites from the Thai-Myanmar nor the Thai-Cambodia border was identified.

In conclusion, the present study confirms the involvement of the pfmdr1 gene in QN sensitivity. Both N86Y and N1042D mutations significantly modulate in vitro response to QN. Although previous studies from Southeast Asia including Vietnam and China-Myanmar border have demonstrated that the pfnhe-1 gene is involved in QN sensitivity, the present study showed no association. This has raised doubt regarding the pfnhe-1 gene as to whether it could be used as the suitable marker for QN resistance in Thailand.