Falciparum Chloroquine Resistance Transporter Gene Research Articles

Epidemiology of drug‐resistant malaria in Republic of Congo: using molecular evidence for monitoring antimalarial drug resistance combined with assessment of antimalarial drug use

In Congo, urgent efforts are needed to help with the revision of the national antimalarial drug policy. Despite its high resistance level, chloroquine (CQ) is still extensively used as the first-line treatment for uncomplicated Plasmodium falciparum malaria. The study was conducted in children under 5 years with uncomplicated malaria in Pointe-Noire and Brazzaville, the two largest cities that contain approximately 60% of the population of Congo. We investigated by polymerized chain reaction and sequencing methods the frequency distribution of molecular markers for antimalarial drug resistance, including mutations in P. falciparum chloroquine resistance transporter (pfcrt) gene associated with CQ resistance and mutations in dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes conferring resistance to sulphadoxine/pyrimethamine (SP) among pre-treatment P. falciparum isolates, as well as assessing antimalarial drug use in the community. pfcrt (K76T) mutation was present in most isolates (96.4%, n = 138) and high frequency (69.2%, n = 133) of triple-mutant dhfr-S108N, N51I, C59R was observed. The quintuple mutant (dhfr-S108N, N51I, C59R and dhps-A437G or S436A, K540E) considered as molecular marker for SP treatment failure was not found because dhps-K540E mutation was absent in isolates tested; this is a clear evidence for the excellent efficacy of SP that we previously described in the same population. The complete absence of the dhps-K540E mutation is a deterrent component for using this molecular marker as an early warning tool for SP resistance testing in that population. Poor compliance issues related to the antimalarial drug use including inappropriate manufacturing practices reported in this study require intensive attention and should be taken into account when implementing drug policy change. If Congo changes its treatment policy from CQ to SP monotherapy, this will not last long. The strategy of combining SP with other affordable and effective antimalarial drugs such as the artemisinin derivatives to improve efficacy and to delay the development of parasite resistance is essential.

Concurrence of Plasmodium falciparum dhfr and crt mutations in northern Ghana

BackgroundBoth chloroquine (CQ) and sulfadoxine-pyrimethamine (SP) are failing drugs in much of sub-Saharan Africa. Previous findings suggest an association between resistance to CQ and to SP in vivo, in vitro, and on the molecular level.MethodsIn 126 Ghanaian children with uncomplicated malaria, associations between mutations conferring resistance in the Plasmodium falciparum dihydrofolate reductase (dhfr; SP) and chloroquine resistance transporter (crt; CQ) genes, concentrations of residual antimalarial drugs, and gametocyte carriage were examined.ResultsMutant dhfr alleles and the CQ-resistance allele crt T76 were strongly associated with each other. Isolates exhibiting the dhfr triple mutation seven times more likely also contained crt T76 parasites as compared to isolates without the dhfr triple variant (P = 0.0001). Moreover, both, isolates with the dhfr triple mutation (adjusted OR, 3.2 (95%CI, 1.0–10.4)) and with crt T76 (adjusted OR, 14.5 (1.4–150.8)) were associated with an increased likelihood of pre-treatment gametocytaemia. However, crt T76 did not correlate with gametocytaemia following SP treatment and no selection of crt T76 in SP treatment failure isolates was observed.ConclusionThese results confirm an association between CQ and SP resistance markers in isolates from northern Ghana. This could indicate accelerated development of resistance to SP if CQ resistance is already present, or vice versa. Considering the enhanced transmission potential as reflected by the increased proportion of isolates containing gametocytes when resistant parasites are present, co-resistance can be expected to spread in this area. However, the underlying mechanism leading to this constellation remains obscure.

ASSOCIATION OF PLASMODIUM FALCIPARUM ISOLATES ENCODING THE P. FALCIPARUM CHLOROQUINE RESISTANCE TRANSPORTER GENE K76T POLYMORPHISM WITH ANEMIA AND SPLENOMEGALY, BUT NOT WITH MULTIPLE INFECTIONS

The aim of the study was to assess whether infections with Plasmodium falciparum isolates encoding the P. falciparum chloroquine resistance transporter (pfcrt) gene K76T polymorphism, a molecular marker for chloroquine resistance, are associated with multiple infections, age, or clinical signs of malaria in a semi-immune population in a holoendemic area of Burkina Faso. The parameters of interest were investigated in 210 P. falciparum-positive inhabitants. Logistic regression analysis showed that pfcrt K76T-carrying isolates are significantly more likely to cause anemia and splenomegaly. Furthermore, we found that infections with P. falciparum isolates encoding pfcrt K76T are dependent on age rather than multiple infections. Our findings suggest that pfcrt K76T might serve as a valuable marker for assessing the long-term clinical effect of chronic infections with chloroquine-resistant P. falciparum isolates in populations, without the need of drug efficacy trials.

Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number

The borders of Thailand harbour the world's most multidrug resistant Plasmodium falciparum parasites. In 1984 mefloquine was introduced as treatment for uncomplicated falciparum malaria, but substantial resistance developed within 6 years. A combination of artesunate with mefloquine now cures more than 95% of acute infections. For both treatment regimens, the underlying mechanisms of resistance are not known. The relation between polymorphisms in the P falciparum multidrug resistant gene 1 (pfmdr1) and the in-vitro and in-vivo responses to mefloquine were assessed in 618 samples from patients with falciparum malaria studied prospectively over 12 years. pfmdr1 copy number was assessed by a robust real-time PCR assay. Single nucleotide polymorphisms of pfmdr1, P falciparum chloroquine resistance transporter gene (pfcrt) and P falciparum Ca2+ ATPase gene (pfATP6) were assessed by PCR-restriction fragment length polymorphism. Increased copy number of pfmdr1 was the most important determinant of in-vitro and in-vivo resistance to mefloquine, and also to reduced artesunate sensitivity in vitro. In a Cox regression model with control for known confounders, increased pfmdr1 copy number was associated with an attributable hazard ratio (AHR) for treatment failure of 6.3 (95% CI 2.9-13.8, p<0.001) after mefloquine monotherapy and 5.4 (2.0-14.6, p=0.001) after artesunate-mefloquine therapy. Single nucleotide polymorphisms in pfmdr1 were associated with increased mefloquine susceptibility in vitro, but not in vivo. Amplification in pfmdr1 is the main cause of resistance to mefloquine in falciparum malaria. Multidrug resistant P falciparum malaria is common in southeast Asia, but difficult to identify and treat. Genes that encode parasite transport proteins maybe involved in export of drugs and so cause resistance. In this study we show that increase in copy number of pfmdr1, a gene encoding a parasite transport protein, is the best overall predictor of treatment failure with mefloquine. Increase in pfmdr1 copy number predicts failure even after chemotherapy with the highly effective combination of mefloquine and 3 days' artesunate. Monitoring of pfmdr1 copy number will be useful in epidemiological surveys of drug resistance in P falciparum, and potentially for predicting treatment failure in individual patients.

SHORT REPORT: POLYMORPHISMS IN THE CHLOROQUINE RESISTANCE TRANSPORTER GENE IN PLASMODIUM FALCIPARUM ISOLATES FROM LOMBOK, INDONESIA

The polymorphisms in the Plasmodium falciparum multidrug resistance 1 (pfmdr1) and P. falciparum chloroquine resistance transporter (pfcrt) genes, which are associated with chloroquine resistance, were examined in 48 P. falciparum isolates from uncomplicated malaria patients from the West Lombok District in Indonesia. The point mutation N86Y in pfmdr1 was present in 35.4% of the isolates and mutation K76T in pfcrt was found in all but one of the samples studied. Identified pfcrt haplotypes were mainly identical to the Papua New Guinea type S(agt)VMNT (42 of 48, 87.5%), and a few isolates had the Southeast Asia type CVIET (5 of 48, 10.4%). Moreover, one P. falciparum isolate harbored the K76N mutation, giving rise to the haplotype CVMNN, which was not previously reported in field isolates. Our findings suggest that chloroquine resistance in this area might have the same origin as in Papua New Guinea.

Molecular markers for drug resistance in malaria: use in treatment, diagnosis and epidemiology.

Malaria and the increasing role of drug resistance as an obstacle to its control are global problems. The identification and implications of molecular markers for antimalarial drug resistance - the subject of this review - are key issues in elucidating and eventually controlling resistance. Recent achievements include the successful expression of the Plasmodium falciparum chloroquine resistance transporter gene, pfcrt, in yeast, the identification of polymorphisms on the gamma-glutamylcysteine synthetase gene, ggcs, as potential determinants of chloroquine and mefloquine resistance, and the usefulness of a combined Plasmodium falciparum dihydrofolate reductase gene, pfdhfr, 59ARG and Plasmodium falciparum dihydropteroate synthase gene, pfdhps, 540GLU marker in reliably representing resistance to antifolates. Moreover, treatment with sulfadoxine-pyrimethamine in the presence of pfdhfr 108ASP alone delayed parasite clearance and increased gametocytogony without an overt loss of the overall therapeutic efficacy of the drug. The use of pfdhfr and pfdhps markers in determining antifolate resistance of Plasmodium falciparum has been consolidated. Similar progress has been made with pfcrt markers for chloroquine resistance, auguring well the operational deployment of molecular techniques. Regarding the molecular basis of resistance to arylaminoalcohols, related drugs, and artemisinin and its derivatives, answers remain elusive, but there are promising new leads.

CLEARANCE OF DRUG-RESISTANT PARASITES AS A MODEL FOR PROTECTIVE IMMUNITY IN PLASMODIUM FALCIPARUM MALARIA

Residents of malaria-endemic areas sometimes spontaneously clear Plasmodium falciparum infection without drug treatment, implying an important role for host factors such as immunity in this clearance. Host factors may also contribute to clearance of parasites resistant to a treatment drug. Chloroquine resistance is caused by point mutations in P. falciparum chloroquine resistance transporter (pfcrt) gene. We investigated the clearance of malaria parasites carrying the key chloroquine resistance-conferring PfCRT mutation K76T in patients treated with chloroquine. We found that the ability to clear these resistant parasites is strongly dependent on age (the best surrogate for protective immunity in endemic areas), suggesting that host immunity plays a critical role in the clearance of resistant P. falciparum infections. Age-adjusted comparison of subjects able to clear resistant parasites and those unable to do so provides a new phenotype for identifying host immune and genetic factors responsible for protective immunity against malaria.

SHORT REPORT: ASSOCIATION BETWEEN CHLOROQUINE AND AMODIAQUINE RESISTANCE AND ALLELIC VARIATION IN THE PLASMODIUM FALCIPARUM MULTIPLE DRUG RESISTANCE 1 GENE AND THE CHLOROQUINE RESISTANCE TRANSPORTER GENE IN ISOLATES FROM THE UPPER NILE IN SOUTHERN SUDAN

Amodiaquine, a 4-aminoquinoline compound, is being considered as an alternative to chloroquine and pyrimethamine/sulfadoxine where resistance in Plasmodium falciparum to both drugs has been selected. Although amodiaquine is more potent than chloroquine, its effectiveness is reduced in areas where chloroquine resistance is high. We report an association of the P. falciparum chloroquine resistance transporter (pfcrt) gene and the P. falciparum multiple drug resistance 1 (pfmdr1) gene, two chloroquine resistance markers, with chloroquine and amodiaquine efficacy in vivo in southern Sudan. The data show that the allele of the pfcrt gene with a lysine to threonine change at codon 76 is strongly associated with both chloroquine and amodiaquine resistance. No such association was observed with the pfmdr1 gene.

RECOVERY OF CHLOROQUINE SENSITIVITY AND LOW PREVALENCE OF THE PLASMODIUM FALCIPARUM CHLOROQUINE RESISTANCE TRANSPORTER GENE MUTATION K76T FOLLOWING THE DISCONTINUANCE OF CHLOROQUINE USE IN MALAWI

In 1993, Malawi stopped treating patients with chloroquine for Plasmodium falciparum malaria because of a high treatment failure rate (58%). In 1998, the in vitro resistance rate to chloroquine was 3% in the Salima District of Malawi; in 2000, the in vivo resistance rate was 9%. We assayed two genetic mutations implicated in chloroquine resistance (N86Y in the P. falciparum multiple drug resistance gene 1 and K76T in the P. falciparum chloroquine resistance transporter gene) in 82 P. falciparum isolates collected during studies in 1998 and 2000. The prevalence of N86Y remained similar to that in neighboring African countries that continued to use chloroquine. In contrast, the prevalence of K76T was substantially lower than in neighboring countries, decreasing significantly from 17% in 1998 to 2% in 2000 (P < 0.02). However, neither mutation was significantly associated with in vivo or in vitro resistance (P > 0.29). Withdrawal of the use of chloroquine appears to have resulted in the recovery of chloroquine efficacy and a reduction in the prevalence of K76T. However, other polymorphisms are also expected to contribute to resistance.

NEW HAPLOTYPES OF THE PLASMODIUM FALCIPARUM CHLOROQUINE RESISTANCE TRANSPORTER (PFCRT) GENE AMONG CHLOROQUINE-RESISTANT PARASITE ISOLATES

Mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene were examined to assess their associations with chloroquine resistance in clinical samples from Armopa (Papua) and Papua New Guinea. In Papua, two of the five pfcrt haplotypes found were new: SVIET from Armopa and CVIKT from an isolate in Timika. There was also a strong association (P < 0.0001) between the pfcrt 76T allele and chloroquine resistance in 50 samples. In Papua New Guinea, mutations in the pfcrt gene were observed in 15 isolates with chloroquine minimum inhibitory concentrations (MICs) of 16-64 pmol, while the remaining six isolates, which had a wild-type pfcrt gene at codon 76, had MICs of 2-8 pmol. These observations confirm that mutations at codon 76 in the pfcrt gene are present in both in vivo and in vitro cases of chloroquine resistance, and that detection of the pfcrt 76T allele could predict potential chloroquine treatment failures.

Plasmodium falciparum pfcrt and pfmdr1 polymorphisms are associated with the pfdhfr N108 pyrimethamine-resistance mutation in isolates from Ghana.

The Plasmodium falciparum chloroquine resistance transporter gene (pfcrt) T76 and multidrug resistance gene analogue (pfmdr1) Y86 mutations are associated with chloroquine(CQ)-resistance. In isolates from 172 pregnant women living in the area of Agogo, Ghana, pfcrt T76 was detected in 69% and pfmdr1 Y86 in 66%. Pfcrt T76 but not pfmdr1 Y86 was more prevalent in samples from women with residual CQ in urine or serum. Parasite densities and multiplicity of infection of pfmdr wild type but not of resistant isolates were reduced by CQ. Adjusted for CQ and pyrimethamine (PYR) in urine, the P. falciparum dihydrofolate reductase (pfdhfr) N108 mutation which confers PYR-resistance was 3.1 and 3 times, respectively, more likely to be detected in isolates containing pfcrt and pfmdr1 mutations than in those comprising wild type alleles. Pfcrt, pfmdr, and pfdhfr mutations are frequent in P. falciparum from this part of Ghana which may limit the choice of drugs for the prevention of malaria in pregnancy. The association of CQ- and PYR-resistance mutations independent of recent drug use could indicate accelerated development of resistance to structurally unrelated drugs. Alternatively, it may reflect selection of resistance in persisting infections due to no longer detectable drug pressure.

High-level chloroquine resistance in Sudanese isolates of Plasmodium falciparum is associated with mutations in the chloroquine resistance transporter gene pfcrt and the multidrug resistance Gene pfmdr1.

Polymorphisms were examined in 2 Plasmodium falciparum genes, as were chloroquine responses of clones and isolates from a village in eastern Sudan. There was a significant association between an allele of the P. falciparum chloroquine resistance transporter gene (pfcrt-T76) and both in vitro and in vivo resistance. There was a less significant association with the multidrug resistance gene pfmdr1-Y86 allele. A significant association between pfmdr1-Y86 and pfcrt-T76 was apparent among resistant isolates, which suggests a joint action of the 2 genes in high-level chloroquine resistance.