Prevalence of pfmdr1, pfcrt, pfdhfr and pfdhps mutations associated with drug resistance, in Luanda, Angola.

BACKGROUND: Malaria is the infectious disease causing the highest morbidity and mortality in Angola and due to widespread chloroquine (CQ) resistance, the country has recently changed its first-line treatment recommendations for uncomplicated malaria, from CQ to artemisinin combination therapies (ACT) in adults, and sulphadoxine/pyrimethamine (S/P) in pregnant women. Loss of SP sensitivity is, however, progressing rapidly in Africa and, in this study, were investigated a number of molecular markers associated to CQ and S/P.
METHODS: Blood samples were collected from 245 children with uncomplicated malaria, admitted at the Pediatric Hospital Dr. David Bernardino (HPDB), Angola, and the occurrence of mutations in Plasmodium falciparum was investigated in the pfmdr1 (N86Y) and pfcrt (K76T) genes, associated with CQ resistance, as well as in pfdhfr (C59R) and pfdhps (K540E), conferring SP resistance.
RESULTS: The frequencies of pfmdr1 mutations in codon 86 were 28.6% N, 61.3% Y and 10.1% mixed infections (NY). The frequency of pfcrt mutations in codon 76 were 93.9% K, 5.7% T and 0.4% mixed infections (KT). For pfdhfr the results were in codon 59, 60.6% C, 20.6% R and 18.8% mixed infections (CR). Concerning pfdhps, 6.3% of the isolates were bearers of the mutation 540E and 5.4% mixed infections (K540E).
CONCLUSION: The results of this epidemiologic study showed high presence of CQ resistance markers while for SP a much lower prevalence was detected for the markers under study.

Background

<span class="Disease">Malaria is endemic throughout much of the Angolan territory, and is by far the highest cause of morbidity and mortality particularly among <span class="Species">children under five years old and pregnant women. Malaria continues to be responsible for 50% of all outpatient attendance and around 25% of all hospital deaths . Recently and according to the Angola Malaria Indicator Survey, 2007, these figures have been reduced due to control programmes, but about 35% of all cases and 70% of all deaths reported annually occur in children under five years of age.

The first cases of resistance to n class="Chemical">chloroquinen> (CQ) were described in the eighties [1-4]. As a consequence of increasing rates of clinical resistance to CQ, the protocol at the Pediatric Hospital David Bernardino (HPDB) was changed, in 2006, two combined therapies artesunate with lumefantrine (Coartem®), and amodiaquine (AQ) with sulphadoxine-pyrimethamine (SP – Fansidar®) as first-line treatments of uncomplicated malaria.

The n class="Gene">pfmdr1n> gene (multidrug resistance) has been described as associated with CQ resistance [5-7], with a polymorphism resulting from the substitution of an asparagine for a tyrosine in amino acid 86 (N86Y) in Plasmodium falciparum [8-14]. CQ resistance is also associated with a mutation in the transporter gene pfcrt where the amino acid substitution at pfcrt codon 76 (K to T) has been shown to have a determinant association with the resistance phenotype [15-19].

<span class="Species">Plasmodium falciparum resistance to <span class="Chemical">sulphadoxine and pyrimethamine (SP) is conferred by mutations of the dihydropteroate synthase (pfdhps) and dihydrofolate reductase (pfdhfr) genes, respectively [20-23]. pfdhfr 108N mutation seems to be enough to confer resistance to pyrimethamine [24,25]. The presence of a mutation at positions 51 (N51I) or 59 (C59R), together with S108N confer a considerable increase in the resistance level to pyrimethamine when compared with mutation S108N by itself [26-28].

Mutations in codons 437 (<span class="Mutation">A437G) and 540 (<span class="Mutation">K540E) of P. falciparum pfdhps are associated with resistance to sulphadoxine. The quintuple mutant (triple pfdhfr: 51I, 59R, 108N and double pfdhps: 437G, 540E) is considered as the molecular marker of SP treatment failure [29-34]. Studies of genetic transfection of P. falciparum, confirmed that the amino acid substitution at pfdhfr codon 108 (S→N), increases approximately ten times the resistance to pyrimethamine [35]. Epidemiology studies also demonstrated that the presence of mutations 59R in pfdhfr and 540E in pfdhps are significantly associated to resistance as well as to the presence of the other above mentioned mutations [30,36,37].

In this work were investigated the frequencies of mutations associated with n class="Chemical">chloroquinen> resistance (pfmdr1 N86Y and pfcrt K76T) and also screened for the two mutations associated to the resistance of P. falciparum to SP as used in populations from high malaria transmission areas.

Materials and methods

Sample collection

Blood samples included in this study were collected from n class="Species">childrenn> between 1 and 16 years, at the HPDB Hospital in Luanda, Angola, and individually spotted on Whatman n.°4 filter paper, after microscopic confirmation of P. falciparum infection. Parent's informed consent was obtained before inclusion of the blood samples in the study which was reviewed and approved by the Ethical Committees from the Ministry of Health of Angola and of the HPDB. Original study was associated to quinine treatment and published elsewhere [38].

Genetic characterization of the parasites

Parasite DNA was extracted from dried blood spots, using Chelex as described elsewhere [39]. <n class="Chemical">span class="Species">P. falciparum mutations associated with resistance to CQ and SP were typed by PCR-RFLP as described elsewhere [39,40], primers sequences, amplification cycles and restrictions enzymes are described in table 1 and 2, respn>ectively. In this study, the following codons and polymorphisms were analysed: pfmdr1 86, pfcrt 76; pfdhfr 59 and pfdhps 540. Amplicons and fragments were separated on 2% or 3% agarose gels stained with ethidium bromide and visualized under UV.

Table 1

PCR Programme for the genes pfmdr1 86, pfcrt 76, pfdhps 540, pfdhfr 59, primer and product size

Gene	Primers	PCR Programme			Product size (bp)
Pfmdr1 86	754N 754R	Initial step	92°C	3 min	321
		Denaturation (35×)	92°C	1 min
		Annealing (35×)	51°C	30 s
		Extension (35×)	72°C	1 min
		Final step	72°C	3 min
Pfcrt 76 1° nested	76o1F N1R	Initial step	94°C	3 min	528
		Denaturation (40×)	94°C	45 s
		Annealing (40×)	55°C	45 s
		Extension (40×)	72°C	45 s
		Final step	72°C	3 min
Pfcrt 76 2° nested	76 N2F 76 N2R	Initial step	94°C	3 min	271
		Denaturation (35×)	94°C	45 s
		Annealing (35×)	53°C	45 s
		Extension (35×)	72°C	45 s
		Final step	72°C	3 min
Pfdhps 540	540 F 540 R	Initial step	94°C	3 min	439
		Denaturation (40×)	94°C	1 min
		Annealing (40×)	45°C	1 min
		Extension (40×)	72°C	1 min
		Final step	72°C	3 min
Pfdhfr 59	59 F 59 R	Initial step	94°C	3 min	326
		Denaturation (35×)	94°C	1 min
		Annealing (35×)	55°C	1 min
		Extension (35×)	72°C	1 min
		Final step	72°C	3 min

Table 2

Fragments length, clones and enzymes used for digestion of the codons 86 Pfmdr 1, 76 Pfcrt, 540 Pfdhps and 59 Pfdhfr.

Gene	Enzyme	clone	Codon	Fragments length
Pfmdr1 86	ApoI	3D7	AAT (Asn)	249 + 72 bp
		Dd2	TAT (Tyr)	321 bp
Pfcrt 76	ApoI	3D7	AAA (Lys)	137 + 124 + 10 bp
		K1	ACA (Thr)	261 + 10 bp
Pfdhps 540	BseGI	N3	GAA (Glu)	354 + 85 bp
		T9/94	AAA (Lys)	439 bp
Pfdhfr 59	PdmI	K1	CGT (Arg)	354 + 85 bp
		HB3	TGT (Cys)	439 bp

PCR Programme for the genes <span class="Gene">pfmdr1n> 86, <span class="Chemical">pfcrt 76, pfdhps 540, pfdhfr 59, primer and product size

Fragments length, clones and enzymes used for digestion of the codons 86 Pfmdr 1, 76 <span class="Chemical">Pfcrtn>, 540 Pfdhps and 59 Pfdhfr.

Statistical analysis

Associations between the different mutations were tested by Fisher's exact test.

Results

Patients and parasites

From the 245 isolates of <span class="Species">P. falciparum parasites used in this study, 199 samples were successfully typed by PCR-RFLP for <span class="Gene">pfmdr1, for pfcrt 245, for pfdhps 221 and for pfdhfr 224. Mutant alleles were detected in four loci: pfcrt 76T, pfmdr1 86Y, pfdhps 540E and pfdhfr 59R (Figure 1).

Figure 1

Agarose gels showing pfmdr1: 2-N86; 3-Y86; 4-N/Y86; 5-3D7 (N86); 6-Dd2 (Y86). pfcrt: 2-k76; 3-T76; 4-K/T76; 5-K1 (K76); 6-3D7 (T76). pfdhfr: 2-C59; 3-R59; 4-R/C59; 5-N3 (C59); 6-T9/94 (R59). pfdhps: 2-E540; 3-K540; 4-K/E540; 5-HB3 (E540); 6-K1 (K540). In all gels lanes 1 and 7 shows the molecular weight marker.

<span class="Chemical">Agarose gels showing <span class="Gene">pfmdr1: 2-N86; 3-Y86; 4-N/Y86; 5-3D7 (N86); 6-Dd2 (Y86). pfcrt: 2-k76; 3-T76; 4-K/T76; 5-K1 (K76); 6-3D7 (T76). pfdhfr: 2-C59; 3-R59; 4-R/C59; 5-N3 (C59); 6-T9/94 (R59). pfdhps: 2-E540; 3-K540; 4-K/E540; 5-HB3 (E540); 6-K1 (K540). In all gels lanes 1 and 7 shows the molecular weight marker.

pfmdr1 and pfcrt

As shown in Table 3, the frequency of the pure mutant allele <span class="Chemical">pfcrt T76 was 93.9%. Only one isolate carried a mixed population KT for this allele (0.41%). The frequency of the mutant <span class="Gene">pfmdr1 Y86 allele was 61.3% and mixed <span class="Gene">pfmdr1 N and Y allele was detected in 10.1% of the isolates.

Table 3

Prevalence of mutations conferring resistance to chloroquine and sulphadoxine/pyrimethamine in Plasmodium falciparum isolates from Angola

Gene	Mutation	n	Mutation (%)	Mixed* (%)
Pfcrt	T76	245	93.9 (230/245)	0.4 (1/245)
pfmdr1	Y86	199	61.3 (122/199)	10.1 (20/199)
pfdhfr	R59	224	20.6 (46/224)	18.8 (42/224)
pfdhps	E540	221	6.3 (14/221)	5.4 (12/221)

*refers to mixed populations of P. falciparum parasites.

Prevalence of mutations conferring resistance to n class="Chemical">chloroquinen> and sulphadoxine/pyrimethamine in Plasmodium falciparum isolates from Angola

*refers to mixed populations of <span class="Species">P. falciparum parasites.

To study the associations between the loci <span class="Chemical">pfcrt <span class="Mutation">K76T and pfmdr1 N86Y, 176 single infection isolates were analysed. Among the 165 isolates with the pfcrt T76 mutation, 115 (70%) also carried the mutant pfmdr1 Y86 allele. Whether or not the mixed infections were excluded from the analysis, the association between the two mutant alleles was significant (P = 0.029, mixed infections excluded).

pfdhps and pfdhfr

One locus of pfdhps (<span class="Mutation">K540E) and one of pfdhfr (<span class="Mutation">C59R) were investigated (Table 3). For the pfdhps 88.3% of the isolates carried the mutant allele E540 and 5.4% carried mixed (K and E) alleles. For pfdhfr 60.6% were wild type (C59) and 18.8% were mixed populations. To study the associations between the pfdhps E540 and pfdhfr R59 loci, only 61 single infection isolates were analysed, and there was no association between pfdhps E540 and pfdhfr R59 (P = 0.159).

Discussion

In vivo resistance to CQ and n class="Chemical">SP have reached high levels in some regions of Angola, where resistance to these drugs is high (25%) [41]. This study reveals a high frequency of drug resistance molecular markers for, CQ (<span class="Chemical">pfcrt T76; 93.9% and <span class="Gene">pfmdr1 Y86; 61.3%) and SP (9% of bearers with quintuple mutant).

In this study, associations within and between the mutations that confer resistance against CQ and those considered predictive of <n class="Chemical">span class="Disease">SP treatment failure were evaluated. Obtained data revealed an association (P = 0.029) between <spn>an class="Chemical">pfcrt T76 (chromosome 7) and pfmdr1 Y86 (chromosome 5) as reported by other authors [42-45].

Presence of mutations R59 and E540 in pfdhfr and pfdhps, respectively, is considered a marker of confirmed resistance to <n class="Chemical">span class="Gene">SP and our data indicates that 9% of the children attending the HPDB, were bearers of P. falciparum parasites with the SP quintuple mutant associated usually to treatment failure. These results are in line with others from western Africa, where low prevalence of the quintuple mutant has been observed [46,47]. Although the predictive value of these markers for SP treatment failure has not been established in this study, these results emphasize the need for close monitoring of mutation prevalence with treatment outcome, since these antimalarials are now used as first line treatment of uncomplicated malaria in children attending the HPDB as well as prophylactic treatment in pregnant women.

Inter- and intragenic association of pfdhfr and pfdhps mutant codons was indirectly proven in other studies where SP resistance was found to be associated with double up to quintuple mutations in both genes [48,49]. Here, only 9% of the samples, with concurrence of the pfdhfr and pfdhps variants, R59 and E450 simultaneously were observed, which are considered to be predictive of the quintuple mutant (pfdhfr I51, R59, N108, pfdhps G437, E540) associated to <n class="Chemical">span class="Disease">SP treatment failure [37,50-53]. All other samples had presence of one or the other mutation suggesting that fixation of the quintuple mutant was still an on going process.

Association between mutations on n class="Chemical">pfcrtn>, pfmdr1, pfdhfr and pfdhps, have been reported from West Africa [54,55] but reports on linkage between response to treatment with CQ and SP, in the same patients, is rare. Even though no statistic association was found, 43 double mutants for pfdhfr/pfcrt, 21 for pfdhfr/pfmdr1, 12 pfdhps/pfcrt and 6 pfdhps/pfmdr1 were detected. Regarding the association between mutations conferring resistance to SP and CQ; almost all of the isolates carrying the mutant genotype for pfdhfr (43 out of 46) or pfdhps (12 out of 14) carried at least one of the CQ associated mutations in the genes pfcrt or pfmdr1. This reflects the possible association and accumulation of at least three or four out of 7 mutations (pfcrt T76, pfmdr1 Y86, pfdhfr: N108, I51, R59 and pfdhps: G437, E540) scattered on four different chromosomes and involved in resistance to three different antimalarials, chloroquine, sulfadoxine and pyrimethamine.

As a result of this study it is not possible to comment whether the presence of n class="Chemical">pfcrtn> T76 favors the presence of pfdhfr and pfdhps mutations or vice versa because this requires longitudinal and long lasting studies and observations. Despite these difficulties in drawing firm conclusions, several previous findings support the hypothesis of linkage disequilibrium between mutations associated with SP resistance and CQ resistance. In a murine malaria model, CQ resistance could be induced in pyrimethamine-resistant parasites, but not in sensitive ones [56].

In field isolates, several observations indicate that it is more likely to find mutations in pfdhfr and pfdhps in isolates exhibiting already <span class="Chemical">pfcrtn> T76 mutation than in isolates comprising <span class="Chemical">pfcrt wild type parasites [57-59]. The reason for this apparent association between resistance to CQ and SP is obscure since both drugs have distinct modes of action and resistance to these is determined by mutations on different chromosomes [60-62]. The accelerated acquisition of resistance to multiple drugs (ARMD) possibly reflecting a rapid mutator phenotype [63] could be one explanation.

Multiple CQ and SP resistance mutations are thought to have higher <span class="Disease">fitness cost of the parasite asexual reproduction (reviewed in [64,65]). In line with the previous considerations, the reproductive capacity of mutant strains is believed to be jeopardized [64-66]. The results did not reflect these since gametocyte prevalence in infections with mutant-type parasites, was not higher than in infections with wild-type isolates.

Conclusion

This was the first molecular study carried out in this geographical area including a considerable number of samples (245) and focused in the mutations of n class="Gene">pfmdr1n>, pfcrt, pfdhps and pfdhfr genes, strongly associated to CQ and SP resistance. The results of the epidemiological study on prevalence of genotypes associated with drug resistance, carried out at a HPDB in Luanda, showed high presence of CQ resistance markers, while for SP a much lower prevalence was detected. This work can be important to evaluate the implementation of new therapeutics strategies based on combinations that includes SP, like the protocol that are now implemented in HPDB (artesunate combined with SP) as first-line drug for uncomplicated malaria treatment.

Competing interests

The authors declare that they have no competing interests

Authors' contributions

PF carried out the molecular analyses and drafted this manuscript. FN participated in the design of the study performed the statistical analysis and helped to draft the manuscript. CB carried out the selection of <span class="Species">childrenn> and sample collection. DL helped in molecular analysis and the draft of the manuscript. LB coordinated sample collection in PHDB as hospital director. VEdR helped in coordination and design of the study. LV coordinated the project and designing of the study. All authors read and approved the final manuscript.