Levels Of Chloroquine Resistance Research Articles

Long cryopreserved lab-adapted Plasmodium falciparum increases resistance to chloroquine but not its susceptibility

Introduction: Plasmodium falciparum (P. falciparum) is a deadly protozoan that is accountable for malaria and chloroquine was the first-line antimalarial drug before its withdrawal and replaced by artemisinin. To date, several studies showed that P. falciparum had regained its sensitivity towards chloroquine after its withdrawal for decades. By understanding the basic principle and mechanism of chloroquine resistance in P. falciparum, at the molecular level, it would be valuable prior to the re-introduction of chloroquine as a first-line anti-malarial drug for malaria treatment. Thus, this study was conducted to determine the chloroquine resistance level of long preserved lab-adapted P. falciparum strain. Methodology: By using 14 years (2006-2020) cryopreserved chloroquine-sensitive (3D7) and chloroquine-resistant (W2) lab-adapted P. falciparum strains, the strains were subjected to continuous culture for three months before in vitro drug susceptibility assay and single nucleotide polymorphisms (SNP) analysis on Pfcrt and Pfmdr-1 gene for both strains. Results: This study shows the IC50 chloroquine of lab-adapted P. falciparum 3D7 and W2 strains were at 32.98 nM and 691.21 nM, respectively and both strains showed 3-fold higher IC50 when compared to their susceptibility before cryopreserved (3D7; 13.84nM and W2; 208.27 nM). The SNPs result showed a consistent amino acid substitution at position 76 (K to T) on PfCRT and 86 (N to Y) in Pfmdr-1 gene which concordance with other studies before preservation. Conclusion: Thus, this study shows that long cryopreserved of lab-adapted P. falciparum increases the chloroquine resistance level but not exhibited any change in susceptibility.

Molecular surveillance of anti-malarial drug resistance in Democratic Republic of Congo: high variability of chloroquinoresistance and lack of amodiaquinoresistance

BackgroundThe loss of chloroquine (CQ) effectiveness has led to its withdrawal from national policies as a first-line treatment for uncomplicated malaria in several endemic countries, such as the Democratic Republic of Congo (DRC). The K76T mutation on the pfcrt gene has been identified as a marker of CQ resistance and the SVMNT haplotype in codons 72–76 on the same gene has been associated with resistance to amodiaquine (AQ). In the DRC, the prevalence of K76T has decreased from 100% in 2000 to 63.9% in 2014. The purpose of this study was to determine the prevalence of K76T mutations in circulating strains of Plasmodium falciparum, 16 years after CQ withdrawal in the DRC and to investigate the presence of the SVMNT haplotype.MethodsIn 2017, ten geographical sites across the DRC were selected. Dried blood samples were collected from patients attending health centres. Malaria was first detected by a rapid diagnostic test (RDT) available on site (SD Bioline Malaria Ag Pf or CareStart Malaria Pf) or thick blood smear and then confirmed by a P. falciparum species-specific real-time PCR assay. A pfcrt gene segment containing a fragment that encodes amino acids at positions 72–76 was amplified by conventional PCR before sequencing.ResultsA total of 1070 patients were enrolled. Of the 806 PCR-confirmed P. falciparum positive samples, 764 were successfully sequenced. The K76T mutation was detected in 218 samples (28.5%; 95% CI 25.4%–31.9%), mainly (96%) with the CVIET haplotype. Prevalence of CQ resistance marker was unequally distributed across the country, ranging from 1.5% in Fungurume to 89.5% in Katana. The SVMNT haplotype, related to AQ resistance, was not detected.ConclusionOverall, the frequency of the P. falciparum CQ resistance marker has decreased significantly and no resistance marker to AQ was detected in the DRC in 2017. However, the between regions variability of CQ resistance remains high in the country. Further studies are needed for continuous monitoring of the CQ resistance level for its prospective re-use in malaria management. The absence of the AQ resistance marker is in line with the use of this drug in the current DRC malaria treatment policy.

Low rates of Plasmodium falciparum Pfcrt K76T mutation in three sentinel sites of malaria monitoring in Côte d'Ivoire.

Despite efforts to eliminate it, malaria remains a major public health concern, particularly in Côte d'Ivoire. Chloroquine (CQ) was one of the first drugs used for its treatment, but was officially withdrawn from the market in 2007 following reports of high levels of chloroquine resistance. The present study was carried out after the withdrawal of CQ and provides an update on the rates of CQ resistance in Côte d'Ivoire. Samples were collected between September 2013 and March 2014 in Abidjan and from January to May 2016 in Abengourou and San Pedro through cross-sectional studies. Parasitemia was assessed by microscopy, and single nucleotide polymorphism in the Pfcrt (codon 76) gene was analyzed by nested PCR and restriction fragment length polymorphism. A total of 343 samples were analyzed: 119, 106 and 118 were from Abidjan, Abengourou, and San Pedro, respectively. The sex ratio of patients was 0.92. The mean age of patients enrolled was 9.6 years (SD = 10.8). The geometric mean of parasite density was 21,337 parasites/μL (SD = 49,508; range, 2,000-200,000). Molecular analysis revealed 57 K76T mutants (16.6%): 33, 9, and 15 in Abidjan, Abengourou and in San Pedro, respectively. Most of these were found in patients aged ≤15 years (42/57) who had parasitemia greater than 10,000 parasites/μL (40/57). This is the first study conducted in Côte d'Ivoire reporting a decline in Pfcrt K76T mutation rate. Thus, our results indicate the importance of following up on the observed trend also at a national level.

Assessment of the molecular marker of Plasmodium falciparum chloroquine resistance (Pfcrt) in Senegal after several years of chloroquine withdrawal.

As a result of widespread antimalarial drug resistance, all African countries with endemic malaria have, in recent years, changed their malaria treatment policy. In Senegal, the health authorities changed from chloroquine (CQ) to a combination of sulfadoxine–pyrimethamine (SP) plus amodiaquine (AQ) in 2003. Since 2006, the artemisinin combination therapies (ACTs) artemether–lumefantrine (AL) and artesunate plus amodiaquine (AS/AQ) were adopted for uncomplicated malaria treatment. After several years of CQ withdrawal, the current study wished to determine the level of CQ resistance at the molecular level in selected sites in Senegal, because the scientific community is interested in using CQ again. Finger prick blood samples were collected from Plasmodium falciparum-positive children below the age of 10 years (N = 474) during cross-sectional surveys conducted in two study sites in Senegal with different malaria transmission levels. One site is in central Senegal, and the other site is in the southern part of the country. All samples were analyzed for single nucleotide polymorphisms (SNPs) in the P. falciparum CQ resistance transporter gene (Pfcrt; codons 72–76) using polymerase chain reaction (PCR) sequence-specific oligonucleotide probe (SSOP) enzyme-linked immunosorbent assay (ELISA) and real-time PCR methods. In total, the 72- to 76-codon region of Pfcrt was amplified in 449 blood samples (94.7%; 285 and 164 samples from the central and southern sites of Senegal, respectively). In both study areas, the prevalence of the Pfcrt wild-type single CVMNK haplotype was very high; in central Senegal, the prevalence was 70.5% in 2009 and 74.8% in 2010, and in southern Senegal, the prevalence was 65.4% in 2010 and 71.0% in 2011. Comparing data with older studies in Senegal, a sharp decline in the mutant type Pfcrt prevalence is evident: from 65%, 64%, and 59.5% in samples collected from various sites in 2000, 2001, and 2004 to approximately 30% in our study. A similar decrease in mutant type prevalence is noted in other neighboring countries. With the continued development of increased CQ susceptibility in many African countries, it may be possible to reintroduce CQ in the near future in a drug combination; it could possibly be given to non-vulnerable groups, but it demands close monitoring of possible reemergence of CQ resistance development.

Quantitative genome re-sequencing defines multiple mutations conferring chloroquine resistance in rodent malaria

BackgroundDrug resistance in the malaria parasite Plasmodium falciparum severely compromises the treatment and control of malaria. A knowledge of the critical mutations conferring resistance to particular drugs is important in understanding modes of drug action and mechanisms of resistances. They are required to design better therapies and limit drug resistance.A mutation in the gene (pfcrt) encoding a membrane transporter has been identified as a principal determinant of chloroquine resistance in P. falciparum, but we lack a full account of higher level chloroquine resistance. Furthermore, the determinants of resistance in the other major human malaria parasite, P. vivax, are not known. To address these questions, we investigated the genetic basis of chloroquine resistance in an isogenic lineage of rodent malaria parasite P. chabaudi in which high level resistance to chloroquine has been progressively selected under laboratory conditions.ResultsLoci containing the critical genes were mapped by Linkage Group Selection, using a genetic cross between the high-level chloroquine-resistant mutant and a genetically distinct sensitive strain. A novel high-resolution quantitative whole-genome re-sequencing approach was used to reveal three regions of selection on chr11, chr03 and chr02 that appear progressively at increasing drug doses on three chromosomes. Whole-genome sequencing of the chloroquine-resistant parent identified just four point mutations in different genes on these chromosomes. Three mutations are located at the foci of the selection valleys and are therefore predicted to confer different levels of chloroquine resistance. The critical mutation conferring the first level of chloroquine resistance is found in aat1, a putative aminoacid transporter.ConclusionsQuantitative trait loci conferring selectable phenotypes, such as drug resistance, can be mapped directly using progressive genome-wide linkage group selection. Quantitative genome-wide short-read genome resequencing can be used to reveal these signatures of drug selection at high resolution. The identities of three genes (and mutations within them) conferring different levels of chloroquine resistance generate insights regarding the genetic architecture and mechanisms of resistance to chloroquine and other drugs. Importantly, their orthologues may now be evaluated for critical or accessory roles in chloroquine resistance in human malarias P. vivax and P. falciparum.

Antimalarial efficacy of piperaquine-based antimalarial combination therapies: facts and uncertainties

Piperaquine is a bisquinoline antimalarial drug extensively used as monotherapy in China in the 1980s and subsequently included as one of the components of the artemisinin-based combination therapies (ACTs) in the 1990s. Among them, dihydroartemisinin-piperaquine (DHA-PQP) represents a new and extremely promising fixed combination. Several clinical trials have repeatedly shown that DHA-PQP is a safe and highly efficacious therapy against uncomplicated Plasmodium falciparum and the asexual stages of Plasmodium vivax malaria. Studies conducted with this drug have reported cure rates consistently above 95%, with the only exception being a study carried out in Papua New Guinea which reported a high rate of treatment failures. Although it has been hypothesized that such treatment failures could be related to cross-resistance mechanisms between piperaquine and other quinolines or to a reduced susceptibility of parasites to artemisinin derivatives, a critical review of the studies published so far seems to exclude both of these possibilities. Overall, there is now sufficient evidence on the safety and efficacy of the DHA-PQP therapy. Accordingly, the use of this ACT for the treatment of P.falciparum malaria has been recently approved in the EU via a centralized procedure by the European Medicines Agency. Moreover, it is now recommended globally by the World Health Organization as an option for the first-line treatment of uncomplicated malaria.

Prevalence of molecular markers of anti-malarial drug resistance in Plasmodium vivax and Plasmodium falciparum in two districts of Nepal

BackgroundSulphadoxine-pyrimethamine (SP) and chloroquine (CQ) have been used in treatment of falciparum and vivax malaria in Nepal. Recently, resistance to both drugs have necessitated a change towards artemisinin combination therapy (ACT) against Plasmodium falciparum in highly endemic areas. However, SP is still used against P. falciparum infections in low endemic areas while CQ is used in suspected cases in areas with lack of diagnostic facilities. This study examines the prevalence of molecular markers of CQ and SP resistance in P. falciparum and Plasmodium vivax to determine if high levels of in vivo resistance are reflected at molecular level as well.MethodsFinger prick blood samples (n = 189) were collected from malaria positive patients from two high endemic districts and analysed for single nucleotide polymorphisms (SNPs) in the resistance related genes of P. falciparum and P. vivax for CQ (Pfcrt, Pfmdr1, Pvmdr1) and SP (Pfdhfr, Pfdhps, Pvdhfr), using various PCR-based methods.Results and discussionPositive P. vivax and P. falciparum infections were identified by PCR in 92 and 41 samples respectively. However, some of these were negative in subsequent PCRs. Based on a few P. falciparum samples, the molecular level of CQ resistance in P. falciparum was high since nearly all parasites had the Pfcrt mutant haplotypes CVIET (55%) or SVMNT (42%), though frequency of the Pfmdr1 wild type haplotype was relatively low (35%). Molecular level of SP resistance in P. falciparum was found to be high. The most prevalent Pfdhfr haplotype was double mutant CNRNI (91%), while frequency of Pfdhps double mutant SGEAA and AGEAA were 38% and 33% respectively. Combined, the frequency of quadruple mutations (CNRNI-SGEAA/AGEAA) was 63%. Based on P. vivax samples, low CQ and SP resistance were most likely due to low prevalence of Pvmdr1 Y976F mutation (5%) and absence of triple/quadruple mutations in Pvdhfr.ConclusionsBased on the limited number of samples, prevalence of CQ and SP resistance at molecular levels in the population in the study area were determined as high in P. falciparum and low in P. vivax. Therefore, CQ could still be used in the treatment of P. vivax infections, but this remains to be tested in vivo while the change to ACT for P. falciparum seems justified.

Prevalence of the molecular marker of chloroquine resistance ( pfcrt 76 ) in Nigeria 5 years after withdrawal of the drug as first-line antimalarial: A cross-sectional study

Background. In line with the World Health Organization (WHO) guideline on chloroquine (CQ) resistance, CQ was withdrawn as the first-line antimalarial drug in Nigeria in 2005 as a result of widespread resistance. It was expected that its sensitivity and clinical usefulness ould be restored with time. This study therefore aimed to determine the level of CQ resistance in Nigerian children aged less than 60 months. Methods. We monitored the resistance pattern 5 years after withdrawal of CQ, using the pfcrt K76T mutation as a molecular marker for CQ resistance. Results. Of 98 Plasmodium falciparum -positive blood samples, 95 (96.9%) showed the K76T mutation. Twenty-seven (27.6%) of the children had been treated with CQ at home before presentation at the clinic, while 50 (51.0%) had taken other antimalarials. Conclusion. Our results indicate that there is an urgent need to re-evaluate antimalarial drug policy in Nigeria, especially when 27.6% of our study population still use CQ at home despite its withdrawal as first-line antimalarial. This may require effective legislation against the manufacture, importation and use of CQ in Nigeria, if the purpose behind its withdrawal is to be achieved.

Different approaches for delivery of Intermittent Preventive Treatment (IPT) to pregnant women in Burkina Faso

Background In Africa the burden of malaria in pregnancy is highest in rural areas. In many Sub-Saharan African countries, IPTp/SP is being adopted to replace chloroquine (CQ) chemoprophylaxis shown to be inefficacious. Many publications showed a very poor compliance with CQ chemoprophylaxis among pregnant women and might explain the failure of this preventive strategy rather than increased levels of CQ resistance. In this study, we compare three approaches of IPTp/SP delivery to pregnant women in term of improving coverage and compliance. These three approaches are: i) Passive health centre services, ii) Extended delivery outreach services, iii) Community based distribution delivery approach.

Limited genetic variation in the Plasmodium falciparum heme detoxification protein (HDP)

Malaria parasites infecting host red blood cells degrade hemoglobin by detoxifying heme into hemozoin. This conversion of heme to hemozoin is performed by a potent protein called heme detoxification protein (HDP), making HDP an attractive target for antimalarial drug development. We studied the genetic variation in Plasmodium falciparum HDP and also investigated if HDP due to its involvement in the heme detoxification pathway is under any potential chloroquine (CQ) selection pressure. We sequenced the complete HDP gene encompassing three exons and two introns (AT and ATTT repeats in intron 1; AT repeats in intron 2) from five P. falciparum laboratory strains with known CQ sensitivity and 50 field isolates from Venezuela (n=26) and Kenya (n=24), with high levels of CQ resistance. Sequencing revealed two mutations, C41F and F91L in exon 1 and exon 2, respectively. The F41 mutation was present only in the CQ sensitive (CQS) HB3 strain. However, all the isolates harbored the 91L mutation, except for the CQS 3D7 strain. The sequencing of the intron 2 region revealed no variation in the number of AT repeats. In contrast, there was a wide variation in the AT and ATTT repeats in intron 1. Overall with respect to the intron 1 repeats, the Venezuelan isolates (Expected heterozygosity, He=0.685) showed less genetic variation as compared to the Kenyan isolates (He=0.986). Furthermore, we also genotyped the 72–76 codons of the pfcrt gene but did not observe any correlation of the pfcrt CQ resistant genotypes (SVMNT or CVIET) with variation in the HDP, thus indicating HDP not to be under any CQ selection pressure. In conclusion, HDP is a conserved target for future antimalarial development.

Monitoring Plasmodium falciparum chloroquine resistance in Yunnan Province, China, 1981–2006

The emergence and spread of drug resistant malaria parasites are an important factor contributing to the global resurgence of malaria, demonstrating the essence of drug resistance surveillance in endemic areas. In the malarious border regions of Yunnan Province, China, we have selected three study sites to monitor in vitro and in vivo resistance of Plasmodium falciparum parasites to chloroquine (CQ) from 1981 to 2006. In vitro studies using the microtest clearly showed high degree of CQ resistance in the early 1980s, when CQ was replaced by artemether monotherapy for falciparum malaria. In subsequent in vitro surveys performed in the early 1990s and 2003–2004, we found reductions in both the concentrations inhibiting 50% parasite growth (IC 50s) and the percentage of resistant parasites at all study sites, although the degrees of the reduction varied among sites. Even though amodiaquine has never been used in this area, there were consistently high levels of resistance to this drug, confirming crossresistance between CQ and amodiaquine. In vivo clinical studies were consistent with the results of the in vitro assays. The overall rate of resistant clinical cases decreased from 97% in 1981–1983 to 40% in 2005–2006. Collectively, whereas a general trend of reduction in CQ resistance was observed in Yunnan, variations among sites existed in this relatively small area, probably as the result of both geographical heterogeneity of malaria epidemiology in Yunnan and different levels of CQ resistance in neighboring countries.

Situation actuelle et perspectives de la prophylaxie du paludisme chez le voyageur et dans les forces armées

Malaria remains a major public health problem, both for travellers and for the 40,000 French soldiers deployed each year to endemic areas. Epidemiological data show that imported malaria (IM) is on the increase, and that migrants account for more than 60% of malaria cases notified each year in France. The increase in IM among French military personnel is explained by prematurely terminated chemoprophylaxis on return, repeated short missions, and more cases of P. vivax and P. ovale infection. The choice of chemoprophylaxis depends mainly on the level of chloroquine resistance in the country visited. The atovaquone-proguanil combination is well tolerated and only requires 7 days of intake on return from the endemic area. Doxycycline monohydrate is cheaper and better-tolerated than mefloquine, and is thus preferred for French military personnel. However, its short half-life necessitates very good compliance. Chemoprophylaxis should be combined with vector control measures and with personal protection (impregnated bednets, protective clothing, repellents, and indoor insecticide spraying). The need for these measures should be clearly explained before departure, during the stay, and after return.

THERAPEUTIC EFFICACY OF ARTEMETHER-LUMEFANTRINE FOR THE TREATMENT OF UNCOMPLICATED PLASMODIUM FALCIPARUM MALARIA IN BANGLADESH

Bangladesh faces increasing levels of chloroquine resistance, and drug sensitivity to sulfadoxine-pyremethamine is already compromised. Therefore, the Ministry of Health recently changed the national treatment guidelines to artemisinin-based combination therapies. The purpose of this study was to determine the baseline therapeutic efficacy of artemether-lumefantrine used as a six-dose regimen for the treatment of uncomplicated Plasmodium falciparum malaria. Sixty-seven patients were enrolled in the study; the cure rate in a 42-day follow-up after an adjustment by polymerase chain reaction was 94.3%. The treatment led to rapid fever (mean +/- SD = 25.82 +/- 12.14 hours) and parasite (30.36 +/- 19.43 hours) clearance. These data suggest that artemether-lumefantrine is a highly efficacious and well-tolerated treatment for uncomplicated P. falciparum malaria in Bangladesh.

Linkage disequilibrium between two distinct loci in chromosomes 5 and 7 of Plasmodium falciparum and in vivo chloroquine resistance in Southwest Nigeria

Chloroquine (CQ) resistance in Plasmodium falciparum is associated with polymorphisms in loci on pfcrt and pfmdr1 genes. In this study, we determined the association and linkage disequilibrium between in vivo CQ resistance and P. falciparum polymorphisms in pfcrt gene at codon 76 and pfmdr1 gene at codon 86 in isolates obtained from 111 children with acute uncomplicated falciparum malaria in Nigeria. Patients were treated with standard dosage of CQ and followed up for 28 days. Filter paper samples were collected at enrollment and during follow-up for parasites genotypes and identification of pfcrt and pfmdr1 mutations. Association and linkage disequilibrium between mutant pfcrtT76 and pfmdr1Y86 alleles in pretreatment isolates of P. falciparum was determined. Fifty-five out of the 111 patients (49.5%) failed treatment. Single mutant pfcrtT76 or pfmdr1Y86 alleles were found in 55 out of 111 P. falciparum isolates screened at enrollment. Of these 55 isolates, the mutant pfcrtT76 and pfmdr1Y86 alleles were found in 84%. Both mutant pfcrtT76 (p=0.0196) and pfmdr1Y86 (p=0.000042) alleles were associated with in vivo CQ resistance. In addition, the mutant pfcrtT76 (p=0.047) and pfmdr1Y86 (p=0.006) alleles were significantly selected by CQ in patients who failed treatment. Association analysis between paired single alleles at pfcrt and pfmdr1 loci showed a significant association (p=0.0349 and chi(2)=4.45) between the pfcrt T76 allele on chromosome 7 and the pfmdr1Y86 allele on chromosome 5 and that these two mutant alleles were in linkage disequilibrium (p=0.000, D'=0.64, and r(2)=0.28). Considering the high level of CQ resistance and drug use in the study area, the observed linkage disequilibrium between the mutant pfcrtT76 and pfmdr1Y86 alleles is maintained epistatically through directional CQ selective pressure.

Prevalence of in vitro resistance to eleven standard or new antimalarial drugs among Plasmodium falciparum isolates from Pointe-Noire, Republic of the Congo.

We determined the level of in vitro resistance of Plasmodium falciparum parasites to standard antimalarial drugs, such as chloroquine, quinine, amodiaquine, halofantrine, mefloquine, cycloguanil, and pyrimethamine, and to new compounds, such as dihydroartemisinin, doxycycline, atovaquone, and lumefantrine. The in vitro resistance to chloroquine reached 75.5%. Twenty-eight percent of the isolates were intermediate or had reduced susceptibility to quinine. Seventy-six percent and 96% of the tested isolates showed in vitro resistance or intermediate susceptibilities to cycloguanil and pyrimethamine, respectively. Only 2% of the parasites demonstrated in vitro resistance to monodesethylamodiaquine. No resistance was shown with halofantrine, lumefantrine, dihydroartemisinin, or atovaquone. Halofantrine, mefloquine, and lumefantrine demonstrated high correlation. No cross-resistance was identified between responses to monodesethyl-amodiaquine, dihydroartemisinin, atovaquone, and cycloguanil. Since the level of chloroquine resistance in vitro exceed an unacceptable upper limit, high rates of in vitro resistance to pyrimethamine and cycloguanil and diminution of the susceptibility to quinine, antimalarial drugs used in combination, such as amodiaquine, artemisinin derivatives, mefloquine, lumefantrine, or atovaquone, seem to be appropriate alternatives for the first line of treatment of acute, uncomplicated P. falciparum malaria.

Analysis of P-glycoprotein expression in purified parasite plasma membrane and food vacuole from Plasmodium falciparum

A P-glycoprotein homologue (Pgh1) is believed to play a role in modulating levels of chloroquine resistance in Plasmodium falciparum. To study the role of Pgh1 in the mechanism of chloroquine (CQ) resistance, antisera were raised against this protein. There was no direct association between the level of Pgh1 expression and chloroquine sensitivity. We also failed to detect phosphorylation of Pgh1 in the food vacuole (FV), suggesting that other mechanisms regulate the chloroquine-resistant (CQR) phenotype. Therefore, high levels of expression of Pgh1 or phosphorylation of this protein in the FV could not account for CQ sensitivity. In addition, the lack of inhibition of CQ accumulation by anti-Pgh1 antibodies suggests that Pgh1 is not involved as a CQ transporter in the plasma membrane of P. falciparum. Furthermore, resistance reversers do not appear to act at the plasma membrane level.

Chloroquine-treatment failure in northern Ghana: roles ofpfcrtT76 andpfmdr1Y86

Although chloroquine (CQ) monotherapy is now generally inadequate for the treatment of Plasmodium falciparum malaria in northern Ghana--recently, 58% of 225 children failed treatment by day 14--use of the drug continues because of its low cost and wide availability. The risk factors associated with CQ-treatment failure in this region of Africa, including the T76 mutation in the chloroquine resistance transporter (pfcrt) gene and the Y86 mutation in the multidrug resistance (pfmdr1) gene of P. falciparum, have now been investigated, and genotype-failure indices (GFI) have been calculated. Treatment failure was found to be associated with young age, poor nutritional status, pfcrt T76 and pfmdr1 Y86, and early treatment failure (ETF) was also associated with high parasitaemia. The presence and concentration of 'residual' CQ in the blood of patients immediately before they were treated with CQ for the present study appeared to have no effect on outcome. Presence at recruitment of pfcrt T76 or pfmdr1 Y86 or both mutations increased the risk of treatment failure by 3.2-, 2.4- and 4.5-fold, and the risk of ETF by 9.8-, 2.7- and 10.2-fold, respectively. The pfcrt T76 GFI for clinical and all treatment failures were 2.8 and 1.4, respectively. These indices were relatively low in the younger children, those with malnutrition, and those with high parasitaemias when treated. Residual CQ did not affect the GFI substantially. Both pfcrt T76 and, to a lesser extent, pfmdr1 Y86 would be useful tools for the surveillance of CQ resistance in northern Ghana. In the current transition phase to alternative first-line treatment for P. falciparum malaria, it should be possible to provide estimates of the level of CQ resistance by monitoring the prevalences of these mutations.

Prevalence of pfcrt point mutations and level of chloroquine resistance in Plasmodium falciparum isolates from Africa

The development in Plasmodium falciparum of the resistance to chloroquine (CQ) constitutes a public health priority, due to its direct influence in childhood mortality. The molecular basis for CQ resistance (CQR) is still unclear but, recently, a new relevant gene, named pfcrt, with several point mutations was identified in P. falciparum. Two mutations, K76T and A220S, have been considered crucial for CQR in further studies, making the pfcrt a good candidate as determinant for CQR in P. falciparum. To contribute to this topic, we have undertaken a molecular screening on 164 P. falciparum isolates from Africa: 120 isolates were Italian imported malaria cases, 27 and 17 isolates were from a school-children survey from Congo and Tanzania, respectively. In vitro tests (pLDH and WHO-Mark III tests) for CQ sensitivity have been also carried out on 28 plasmodial isolates and results compared to those obtained by molecular analysis in the same isolates. The SVIET pfcrt haplotype has been identified in the samples from Congo, and this is the first time that this haplotype is detected in Africa. Our results give further evidence to the reliability of the 76T (and the linked 74I–75E) pfcrt point mutation as molecular marker for CQR.

COMPARATIVE EFFICACY OF CHLOROQUINE AND SULFADOXINE-PYRIMETHAMINE FOR UNCOMPLICATED PLASMODIUM FALCIPARUM MALARIA AND IMPACT ON GAMETOCYTE CARRIAGE RATES IN THE EAST NUSATENGGARA PROVINCE OF INDONESIA

The efficacy of chloroquine (CQ) and sulfadoxine-pyrimethamine (SP) was evaluated in 89 subjects greater than one year of age with uncomplicated P. falciparum malaria in the East Nusatenggara Province of Indonesia. Fever clearance time was longer in the SP group than in the CQ group. However, parasite clearance time was extended in subjects who received CQ compared with those who received SP. Major adverse events were not observed in either group, and no hospitalizations were required during the study. Treatment failure rates at day 28 were 69% for CQ and 8.5% for SP. In both treatment groups, gametocytemia developed during the follow-up period, but was more pronounced in the SP group, peaking at 94% on day 7. Regardless of treatment group, children < 10 years of age had significantly higher treatment failure rates than subjects >/=10 years of age (relative risk = 2.49), suggesting that acquired immunity influenced treatment outcomes in the presence of parasite drug resistance. Although a highly effective alternative to CQ for clearing infection, SP treatment also presented some potential drawbacks (e.g., increased and persistent gametocytemia). Replacement of CQ with SP as a first-line therapy, either alone or in combination with CQ, in those areas of Indonesia with high levels of CQ resistance should significantly improve treatment outcomes, particularly in vulnerable populations lacking clinical immunity. More efficacious and rapidly acting asexual stage treatments are generally associated with increased gametocyte clearance and combination therapy in areas where drug resistance is high or emerging may provide an additional means for reducing transmission.

Decreasing clinical efficacy of chloroquine in Ankazobe, Central Highlands of Madagascar

The clinical efficacy of chloroquine was assessed in the Primary Health Centre of Ankazobe, Central Highlands of Madagascar. This study shows an increase in the level of chloroquine resistance with the appearance of early treatment failures and RIII resistance. Furthermore, the prevalence of clinical treatment failures is approaching the level of 25%, at which WHO recommends a change of first-line drug.