Tools for surveillance of anti-malarial drug resistance: an assessment of the current landscape.

To limit the spread and impact of anti-malarial drug resistance and react accordingly, surveillance systems able to detect and track in real-time its emergence and spread need to be strengthened or in some places established. Currently, surveillance of anti-malarial drug resistance is done by any of three approaches: (1) in vivo studies to assess the efficacy of drugs in patients; (2) in vitro/ex vivo studies to evaluate parasite susceptibility to the drugs; and/or (3) molecular assays to detect validated gene mutations and/or gene copy number changes that are associated with drug resistance. These methods are complementary, as they evaluate different aspects of resistance; however, standardization of methods, especially for in vitro/ex vivo and molecular techniques, is lacking. The World Health Organization has developed a standard protocol for evaluating the efficacy of anti-malarial drugs, which is used by National Malaria Control Programmes to conduct their therapeutic efficacy studies. Regional networks, such as the East African Network for Monitoring Antimalarial Treatment and the Amazon Network for the Surveillance of Antimalarial Drug Resistance, have been set up to strengthen regional capacities for monitoring anti-malarial drug resistance. The Worldwide Antimalarial Resistance Network has been established to collate and provide global spatial and temporal trends information on the efficacy of anti-malarial drugs and resistance. While exchange of information across endemic countries is essential for monitoring anti-malarial resistance, sustainable funding for the surveillance and networking activities remains challenging. The technology landscape for molecular assays is progressing quite rapidly, and easy-to-use and affordable new techniques are becoming available. They also offer the advantage of high throughput analysis from a simple blood spots obtained from a finger prick. New technologies combined with the strengthening of national reference laboratories in malaria-endemic countries through standardized protocols and training plus the availability of a proficiency testing programme, would contribute to the improvement and sustainability of anti-malarial resistance surveillance networks worldwide.

A Worldwide Map of Plasmodium falciparum K13-Propeller Polymorphisms.

BACKGROUND: Recent gains in reducing the global burden of malaria are threatened by the emergence of Plasmodium falciparum resistance to artemisinins. The discovery that mutations in portions of a P. falciparum gene encoding kelch (K13)-propeller domains are the major determinant of resistance has provided opportunities for monitoring such resistance on a global scale. METHODS: We analyzed the K13-propeller sequence polymorphism in 14,037 samples collected in 59 countries in which malaria is endemic. Most of the samples (84.5%) were obtained from patients who were treated at sentinel sites used for nationwide surveillance of antimalarial resistance. We evaluated the emergence and dissemination of mutations by haplotyping neighboring loci. RESULTS: We identified 108 nonsynonymous K13 mutations, which showed marked geographic disparity in their frequency and distribution. In Asia, 36.5% of the K13 mutations were distributed within two areas--one in Cambodia, Vietnam, and Laos and the other in western Thailand, Myanmar, and China--with no overlap. In Africa, we observed a broad array of rare nonsynonymous mutations that were not associated with delayed parasite clearance. The gene-edited Dd2 transgenic line with the A578S mutation, which expresses the most frequently observed African allele, was found to be susceptible to artemisinin in vitro on a ring-stage survival assay. CONCLUSIONS: No evidence of artemisinin resistance was found outside Southeast Asia and China, where resistance-associated K13 mutations were confined. The common African A578S allele was not associated with clinical or in vitro resistance to artemisinin, and many African mutations appear to be neutral. (Funded by Institut Pasteur Paris and others.).

Performance of Paracheck™-Pf, SD Bioline malaria Ag-Pf and SD Bioline malaria Ag-Pf/pan for diagnosis of falciparum malaria in the Central African Republic.

BACKGROUND: Rapid diagnostic tests (RDTs) are the current complement to microscopy for ensuring prompt malaria treatment. We determined the performance of three candidate RDTs (Paracheck™-Pf, SD Bioline malaria Ag-Pf and SD Bioline malaria Ag-Pf/pan) for rapid diagnosis of malaria in the Central African Republic. METHODS: Blood samples from consecutive febrile patients who attended for laboratory analysis of malaria at the three main health centres of Bangui were screened by microscopy and the RDTs. Two reference standards were used to assess the performance of the RDTs: microscopy and, a combination of microscopy plus nested PCR for slides reported as negative, on the assumption that negative results by microscopy were due to sub-patent parasitaemia. RESULTS: We analysed 436 samples. Using the combined reference standard of microscopy + PCR, the sensitivity of Paracheck™-Pf was 85.7% (95% CI, 80.8-89.8%), that of SD Bioline Ag-Pf was 85.4% (95% CI, 80.5-90.7%), and that of SD Bioline Ag-Pf/pan was 88.2% (95% CI, 83.2-92.0%). The tests performed less well in cases of low parasitaemia; however, the sensitivity was > 95% at > 500 parasites/µl. CONCLUSIONS: Overall, SD Bioline malaria Ag-Pf and SD Bioline malaria Ag-Pf/pan performed slightly better than Paracheck™-Pf. Use of RDTs with reinforced microscopy practice and laboratory quality assurance should improve malaria treatment in the Central African Republic.

Efficacy and safety of artemether + lumefantrine, artesunate + sulphamethoxypyrazine-pyrimethamine and artesunate + amodiaquine and sulphadoxine-pyrimethamine + amodiaquine in the treatment of uncomplicated falciparum malaria in Bangui, Central African Republic: a randomized trial.

BACKGROUND: The efficacy of artemisinin-based combination therapy (ACT) has been established. The objective of the present study was to compare the efficacy and safety in the Central African Republic (CAR) of three commercially available artemisinin-based combinations, artemether + lumefantrine (AL), artesunate + sulphamethoxypyrazine-pyrimethamine (AS-SMP) and artesunate + amodiaquine (AS-AQ), with those of sulphadoxine-pyrimethamine + amodiaquine (SP-AQ), which was the first-line reference treatment in the country from 2004, until it was replaced by ACT in 2006 in accordance with changes in international recommendations based on resistance identified in other regions. METHODS: Children aged six to 59 months with uncomplicated Plasmodium falciparum malaria were recruited in Bangui, the capital of the CAR. The 251 patients selected were randomly assigned to receive AL (n = 60), AS-SMP (n = 58), AS-AQ (n = 68) or SP-AQ (n = 65) and were followed up for 28 days. Clinical outcome was classified according to the standard 2003 World Health Organization protocol. RESULTS: At day 28, the cure rates in a per-protocol analysis were 92% (48/52) with AL, 93% (50/54) with AS-SMP, 93% (55/59) with AS-AQ and 100% (57/57) with SP-AQ, with no statistically significant difference between the four treatments. Defervescence was significantly faster with AS-AQ than with AL (p <0.035). Fatigue was reported significantly more frequently by patients receiving AQ than by those treated with AS-SMP or AL (p = 0.006). All the other adverse events reported were mild, and no significant difference was noted by treatment. CONCLUSION: The three artemisinin-bsed combinations show similar, satisfactory results, comparable to that with SP-AQ. This evaluation is the first conducted in CAR since the official introduction of ACT. It should guide the National Malaria Control Programme in choosing the appropriate ACT for treatment of uncomplicated P. falciparum malaria in the future.

Rational case management of malaria with a rapid diagnostic test, Paracheck Pf®, in antenatal health care in Bangui, Central African Republic.

BACKGROUND: Both treatment and prevention strategies are recommended by the World Health Organization for the control of malaria during pregnancy in tropical areas. The aim of this study was to assess use of a rapid diagnostic test for prompt management of malaria in pregnancy in Bangui, Central African Republic. METHODS: A cohort of 76 pregnant women was screened systematically for malaria with ParacheckPf® at each antenatal visit. The usefulness of the method was analysed by comparing the number of malaria episodes requiring treatment in the cohort with the number of prescriptions received by another group of pregnant women followed-up in routine antenatal care. RESULTS: In the cohort group, the proportion of positive ParacheckPf® episodes during antenatal clinics visits was 13.8%, while episodes of antimalarial prescriptions in the group which was followed-up routinely by antenatal personnel was estimated at 26.3%. Hence, the relative risk of the cohort for being prescribed an antimalarial drug was 0.53. Therefore, the attributable fraction of presumptive treatment avoided by systematic screening with ParacheckPf® was 47%. CONCLUSIONS: Use of a rapid diagnostic test is useful, affordable and easy for adequate treatment of malaria in pregnant women. More powerful studies of the usefulness of introducing the test into antenatal care are needed in all heath centres in the country and in other tropical areas.

Pattern of the Antimalarials Prescription during Pregnancy in Bangui, Central African Republic.

Introduction. The aim of this study was to identify the antimalarials prescribed during the pregnancy and to document their timing. Method. From June to September 2009, a survey was conducted on 565 women who gave birth in the Castors maternity in Bangui. The antenatal clinics cards were checked in order to record the types of antimalarials prescribed during pregnancy according to gestational age. Results. A proportion of 28.8% ANC cards contained at least one antimalarial prescription. The commonest categories of antimalarials prescribed were: quinine (56.7%), artemisinin-based combinations (26.8%) and artemisinin monotherapy (14.4%). Among the prescriptions that occurred in the first trimester of pregnancy, artemisinin-based combinations and artemisinin monotherapies represented the proportions of (10.9%) and (13.3%). respectively. Conclusion. This study showed a relatively high rate (>80%) of the recommended antimalarials prescription regarding categories of indicated antimalarials from national guidelines. But, there is a concern about the prescription of the artemisinin derivatives in the first trimester of pregnancy, and the prescription of artemisinin monotherapy. Thus, the reinforcement of awareness activities of health care providers on the national malaria treatment during pregnancy is suggested.

Relatively low prevalence of peripheral and placental Plasmodium infection at delivery in bangui, central african republic.

Introduction. The aim of this study was to estimate the prevalence of malaria among women giving birth in Bangui. Association between sociodemographic characteristics of those women and malaria, as well as prevention compliance (use of intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTsp) and insecticide-treated bed nets (ITNs)), was analyzed. Methods. During September 2009, a survey was conducted on 328 women who gave birth at two main maternities of Bangui. Information was obtained by standardized questionnaire about sociodemographic criteria, IPTsp, other antimalarial treatment, and use of bet nets. Smears prepared from peripheral and placental blood were analysed for malaria parasites. Findings and Discussion. Positive results were found in 2.8% of thick peripheral blood smears and in 4.0% of placental slides. A proportion of 30.5% of the women had received at least two doses of IPTsp during the current pregnancy. Only a proportion of 42.4% of this study population had ITNs. Multigravid women were less likely to use IPTsp and ITNs. However, use of IPTsp was associated with personal income and secondary or university educational status. Hence, although this relatively prevalence was observed, more efforts are needed to implement IPTsp and ITNs, taking into account sociodemographic criteria.

Global sequence variation in the histidine-rich proteins 2 and 3 of Plasmodium falciparum: implications for the performance of malaria rapid diagnostic tests.

BACKGROUND: Accurate diagnosis is essential for prompt and appropriate treatment of malaria. While rapid diagnostic tests (RDTs) offer great potential to improve malaria diagnosis, the sensitivity of RDTs has been reported to be highly variable. One possible factor contributing to variable test performance is the diversity of parasite antigens. This is of particular concern for Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-detecting RDTs since PfHRP2 has been reported to be highly variable in isolates of the Asia-Pacific region. METHODS: The pfhrp2 exon 2 fragment from 458 isolates of P. falciparum collected from 38 countries was amplified and sequenced. For a subset of 80 isolates, the exon 2 fragment of histidine-rich protein 3 (pfhrp3) was also amplified and sequenced. DNA sequence and statistical analysis of the variation observed in these genes was conducted. The potential impact of the pfhrp2 variation on RDT detection rates was examined by analysing the relationship between sequence characteristics of this gene and the results of the WHO product testing of malaria RDTs: Round 1 (2008), for 34 PfHRP2-detecting RDTs. RESULTS: Sequence analysis revealed extensive variations in the number and arrangement of various repeats encoded by the genes in parasite populations world-wide. However, no statistically robust correlation between gene structure and RDT detection rate for P. falciparum parasites at 200 parasites per microlitre was identified. CONCLUSIONS: The results suggest that despite extreme sequence variation, diversity of PfHRP2 does not appear to be a major cause of RDT sensitivity variation.

Polymorphisms in pfcrt, pfmdr1, dhfr genes and in vitro responses to antimalarials in Plasmodium falciparum isolates from Bangui, Central African Republic.

Drug resistance is probably the greatest challenge to most malaria-control programs. Given the limited resources for other malarial-control measures, rational drug used is crucial. Molecular markers for parasite resistance such as pfcrt, pfmdr-1, and dhfr have the potential to be used in an integrated fashion to provide timely information that is useful to policy makers. Therefore, we evaluated polymorphisms in these genes from Plasmodium falciparum and their association with in vitro antimalarial drug resistance to 135 parasites samples collected in Bangui in 2004. For the dhfr gene, we found a strong association between the dhfr genotype and chemosensitivity to pyrimethamine. For the pfcrt gene, we found that haplotypes with mutant-type alleles led to significant changes in the IC50 values for chloroquine, monodesethylamodiaquine, and quinine. We found no correlations for the pfmdr1 gene. These findings suggest that a regular monitoring and screening for resistance markers for antifolates and for chloroquine could act as an adjunct to in vivo trials.

Frequency distribution of antimalarial drug-resistant alleles among isolates of Plasmodium falciparum in Bangui, Central African Republic.

We determined the baseline frequency distribution of mutant alleles of genes associated with resistance to chloroquine and sulfadoxine-pyrimethamine in Plasmodium falciparum isolates in Bangui, Central African Republic. Mutant alleles of the P. falciparum chloroquine resistance transporter (pfcrt) gene were found in all samples and the frequency of the deduced CIET pfcrt haplotype was high (45%). The most common allele of the P. falciparum multidrug resistance 1 (pfmdr1) gene among the field isolates of P. falciparum was 86Y (21.9%). The 1246Y allele was also common (18.0%). Of the 167 P. falciparum isolates in which the dihydrofolate reductase gene was studied, only 11 carried the wild-type allele (6.6%) whereas many (50.3%) were quadruple mutants (50R, 51I, 59R, 108N). The frequency of the 436A mutant allele of the dihydropteroate synthase gene was high (74.3%), but the frequencies of the 437G (18.6%) and 540E (5.2%) mutant alleles were low. Molecular analyses of antimalarial drug-resistant alleles of P. falciparum isolates in Bangui strongly suggest the widespread distribution of chloroquine and pyrimethamine resistance and to a lesser extent sulfadoxine resistance.

Association of failures of seven-day courses of artesunate in a non-immune population in Bangui, Central African Republic with decreased sensitivity of Plasmodium falciparum.

We assessed the efficacy and safety of a seven-day course of artesunate for the treatment of uncomplicated Plasmodium falciparum malaria in 55 non-immune patients living in Bangui, Central African Republic. The parasitologic cure rates were 100%, 95%, and 85% on days 14, 28, and 42, respectively. There were no significant differences in parasitemia density, 50% inhibitory concentration of dihydroartemisinin, and frequency of mutant P. falciparum multidrug resistance 1 codon 86 between patients who were cured and those who displayed recrudescence. However, the 90% inhibitory concentration for dihydroartemisinin and the number of genotypes isolated were both higher in the recrudescent patients (five- and two-fold, respectively). We found an association between recrudescence and decreased sensitivity. This suggests that the use of artemisinin compounds alone will select resistant strains. We conclude that artesunate should not be used in monotherapy even in seven-day courses, but only in combination with other anti-malarials to prevent the emergence of resistant P. falciparum.

Drug-resistant malaria in Bangui, Central African Republic: an in vitro assessment.

We used an in vitro isotopic drug sensitivity assay to assess the sensitivity of Plasmodium falciparum isolates collected in Bangui, Central African Republic between March and July 2004. We tested antimalarials that are currently in use in this country (chloroquine, amodiaquine, quinine, and pyrimethamine), antimalarials that will become available in this region in the future (artemisinin and halofantrine), and prophylactic antimalarials (mefloquine, doxycycline, and atovaquone). The proportions of resistant isolates were 37% for chloroquine, 15.9% for amodiaquine, 0% for quinine, 0% for dihydroartemisinin, 1.6% for mefloquine, 3.8% for halofantrine, 4.0% for atovaquone, and 38.3% for pyrimethamine. No multi-resistant isolates (showing resistance to more than three drugs) were found. A positive correlation was found between the 50% inhibitory concentrations values for the following drugs: chloroquine and amodiaquine; quinine and halofantrine; chloroquine and dihydroartemisinin; chloroquine and halofantrine; amodiaquine and dihydroartemisinin; dihydroartemisinin and mefloquine; chloroquine and quinine; and quinine and dihydroartemisinin. These findings suggest that the Ministry of Health should recommend a interim policy with the amodiaquine plus sulfadoxine-pyrimethamine combination as the first-line antimalarial drug in Bangui until better alternative treatments such as artemisinin-based combination therapies become available at low prices in the Central African Republic.

Efficacy of chloroquine, amodiaquine, sulfadoxine-pyrimethamine, chloroquine-sulfadoxine-pyrimethamine combination, and amodiaquine-sulfadoxine-pyrimethamine combination in Central African children with noncomplicated malaria.

This paper reports a two-phase study in Bangui, Central African Republic (CAR): first, we assessed the clinical efficacy to chloroquine (CQ), sulfadoxine-pyrimethamine (SP), and amodiaquine (AQ), then we tested the efficacy of two combinations: CQ + SP and AQ + SP. We used the standard 14-day WHO 2001 protocol to compare therapeutic responses in children under 5 years of age with acute uncomplicated Plasmodium falciparum malaria in Bangui between February 2002 and March 2004. The overall treatment failure rates with CQ, AQ, SP, CQ + SP, and AQ + SP were 40.9%, 20.0%, 22.8%, 7.2%, and 0%. These findings suggest that the Ministry of Health should recommend an interim policy with AQ + SP combination as the first-line antimalarial drug in Bangui until best alternative treatments like artemisinin-based combination therapies (ACTs) become available at low prices in the CAR.