Varying efficacy of artesunate+amodiaquine and artesunate+sulphadoxine-pyrimethamine for the treatment of uncomplicated falciparum malaria in the Democratic Republic of Congo: a report of two in-vivo studies

BACKGROUND: Very few data on anti-malarial efficacy are available from the Democratic Republic of Congo (DRC). DRC changed its anti-malarial treatment policy to amodiaquine (AQ) and artesunate (AS) in 2005. METHODS: The results of two in vivo efficacy studies, which tested AQ and sulphadoxine-pyrimethamine (SP) monotherapies and AS+SP and AS+AQ combinations in Boende (Equatorial province), and AS+SP, AS+AQ and SP in Kabalo (Katanga province), between 2003 and 2004 are presented. The methodology followed the WHO 2003 protocol for assessing the efficacy of anti-malarials in areas of high transmission. RESULTS: Out of 394 included patients in Boende, the failure rates on day 28 after PCR-genotyping adjustment of AS+SP and AS+AQ were estimated as 24.6% [95% CI: 16.6-35.5] and 15.1% [95% CI: 8.6-25.7], respectively. For the monotherapies, failure rates were 35.9% [95% CI: 27.0-46.7] for SP and 18.3% [95% CI: 11.6-28.1] for AQ. Out of 207 patients enrolled in Kabalo, the failure rate on day 28 after PCR-genotyping adjustment was 0 [1-sided 95% CI: 5.8] for AS+SP and AS+AQ [1-sided 95% CI: 6.2]. It was 19.6% [95% CI: 11.4-32.7] for SP monotherapy. CONCLUSION: The finding of varying efficacy of the same combinations at two sites in one country highlights one difficulty of implementing a uniform national treatment policy in a large country. The poor efficacy of AS+AQ in Boende should alert the national programme to foci of resistance and emphasizes the need for systems for the prospective monitoring of treatment efficacy at sentinel sites in the country

Optimization and validation of multi-coloured capillary electrophoresis for genotyping of Plasmodium falciparum merozoite surface proteins (msp1 and 2)

BACKGROUND: Genotyping of Plasmodium falciparum based on PCR amplification of the polymorphic genes encoding the merozoite surface proteins 1 and 2 (msp1 and msp2) is well established in the field of malaria research to determine the number and types of concurrent clones in an infection. Genotyping is regarded essential in anti-malarial drug trials to define treatment outcome, by distinguishing recrudescent parasites from new infections. Because of the limitations in specificity and resolution of gel electrophoresis used for fragment analysis in most genotyping assays it became necessary to improve the methodology. An alternative technique for fragment analysis is capillary electrophoresis (CE) performed using automated DNA sequencers. Here, one of the most widely-used protocols for genotyping of P. falciparum msp1 and msp2 has been adapted to the CE technique. The protocol and optimization process as well as the potentials and limitations of the technique in molecular epidemiology studies and anti-malarial drug trials are reported. METHODS: The original genotyping assay was adapted by fluorescent labeling of the msp1 and msp2 allelic type specific primers in the nested PCR and analysis of the final PCR products in a DNA sequencer. A substantial optimization of the fluorescent assay was performed. The CE method was validated using known mixtures of laboratory lines and field samples from Ghana and Tanzania, and compared to the original PCR assay with gel electrophoresis. RESULTS: The CE-based method showed high precision and reproducibility in determining fragment size (< 1 bp). More genotypes were detected in mixtures of laboratory lines and blood samples from malaria infected children, compared to gel electrophoresis. The capacity to distinguish recrudescent parasites from new infections in an anti-malarial drug trial was similar by both methods, resulting in the same outcome classification, however with more precise determination by CE. CONCLUSION: The improved resolution and reproducibility of CE in fragment sizing allows for comparison of alleles between separate runs and determination of allele frequencies in a population. The more detailed characterization of individual msp1 and msp2 genotypes may contribute to improved assessments in anti-malarial drug trials and to a further understanding of the molecular epidemiology of these polymorphic P. falciparum antigens

Development of a TaqMan Allelic Discrimination Assay for detection of Single Nucleotides Polymorphisms associated with anti-malarial drug resistance

<p>Abstract</p> <p>Background</p> <p>Anti-malarial drug resistance poses a threat to current global efforts towards control and elimination of malaria. Several methods are used in monitoring anti-malarial drug resistance. Molecular markers such as single nucleotide polymorphism (SNP) for example are increasingly being used to identify genetic mutations related to anti-malarial drug resistance. Several methods are currently being used in analysis of SNP associated with anti-malarial drug resistance and although each one of these methods has unique strengths and shortcoming, there is still need to improve and/or develop new methods that will close the gap found in the current methods.</p> <p>Methods</p> <p>TaqMan Allelic Discrimination assays for detection of SNPs associated with anti-malarial drug resistance were designed for analysis on Applied Biosystems PCR platform. These assays were designed by submitting SNP sequences associated with anti-malarial drug resistance to Applied Biosystems website. Eleven SNPs associated with resistance to anti-malarial drugs were selected and tested. The performance of each SNP assay was tested by creating plasmid DNAs carrying codons of interests and analysing them for analysis. To test the sensitivity and specificity of each SNP assay, 12 clinical samples were sequenced at codons of interest and used in the analysis. Plasmid DNAs were used to establish the Limit of Detection (LoD) for each assay.</p> <p>Results</p> <p>Data from genetic profiles of the <it>Plasmodium falciparum </it>laboratory strains and sequence data from 12 clinical samples was used as the reference method with which the performance of the SNP assays were compared to. The sensitivity and specificity of each SNP assay was establish at 100%. LoD for each assay was established at 2 GE, equivalent to less than 1 parasite/μL. SNP assays performed well in detecting mixed infection and analysis of clinical samples.</p> <p>Conclusion</p> <p>TaqMan Allelic Discrimination assay provides a good alternative tool in detection of SNPs associated with anti-malarial drug.</p

Therapeutic efficacy of sulphadoxine-pyrimethamine and chloroquine for the treatment of uncomplicated malaria in pregnancy in Burkina Faso

BACKGROUND: A reduction in the therapeutic efficacy of chloroquine (CQ) and sulphadoxine-pyrimethamine (SP) has recently been observed in Burkina Faso. As these two drugs are used in pregnancy, their efficacy in pregnant women was studied to directly assess the level of drug resistance in this specific population, rather than to extrapolate results of studies conducted in children < 5 years of age. METHODS: During the malaria transmission season of 2003 in Ouagadougou, the clinical efficacy of SP and CQ, using the WHO 28-day protocol, was assessed in primigravidae and secundigravidae presenting with uncomplicated malaria. RESULTS: PCR-corrected results by day 28 showed that among 62 women treated with SP, eight (12.9%) experienced late parasitological failure, but no clinical failures. Among 60 women treated with CQ, the overall failure rate was 46.7% including 1.7% early treatment failures, 5% late clinical failures and 40% late parasitological failures. SP induced a haemoglobin gain of 0.3 g/dL by day 14 and 0.9 g/dL by day 28. Treatment responses were independent of gravidity, gestational age and prior antenatal care visits. CONCLUSION: While CQ should no longer be used, the efficacy of SP is still compatible with use for intermittent preventive treatment (IPT) in pregnancy. However, given the possible spread of resistance, the drug should be restricted in its use

Dihydroartemisinin-Piperaquine and Artemether-Lumefantrine for Treating Uncomplicated Malaria in African Children: A Randomised, Non-Inferiority Trial

BACKGROUND: Artemisinin combination therapies (ACTs) are currently the preferred option for treating uncomplicated malaria. Dihydroartemisinin-piperaquine (DHA-PQP) is a promising fixed-dose ACT with limited information on its safety and efficacy in African children. METHODOLOGY/PRINCIPAL FINDINGS: The non-inferiority of DHA-PQP versus artemether-lumefantrine (AL) in children 6-59 months old with uncomplicated P. falciparum malaria was tested in five African countries (Burkina Faso, Kenya, Mozambique, Uganda and Zambia). Patients were randomised (2:1) to receive either DHA-PQP or AL. Non-inferiority was assessed using a margin of -5% for the lower limit of the one-sided 97.5% confidence interval on the treatment difference (DHA-PQP vs. AL) of the day 28 polymerase chain reaction (PCR) corrected cure rate. Efficacy analysis was performed in several populations, and two of them are presented here: intention-to-treat (ITT) and enlarged per-protocol (ePP). 1553 children were randomised, 1039 receiving DHA-PQP and 514 AL. The PCR-corrected day 28 cure rate was 90.4% (ITT) and 94.7% (ePP) in the DHA-PQP group, and 90.0% (ITT) and 95.3% (ePP) in the AL group. The lower limits of the one-sided 97.5% CI of the difference between the two treatments were -2.80% and -2.96%, in the ITT and ePP populations, respectively. In the ITT population, the Kaplan-Meier estimate of the proportion of new infections up to Day 42 was 13.55% (95% CI: 11.35%-15.76%) for DHA-PQP vs 24.00% (95% CI: 20.11%-27.88%) for AL (p<0.0001). CONCLUSIONS/SIGNIFICANCE: DHA-PQP is as efficacious as AL in treating uncomplicated malaria in African children from different endemicity settings, and shows a comparable safety profile. The occurrence of new infections within the 42-day follow up was significantly lower in the DHA-PQP group, indicating a longer post-treatment prophylactic effect. TRIAL REGISTRATION: Controlled-trials.com ISRCTN16263443

Host candidate gene polymorphisms and clearance of drug-resistant Plasmodium falciparum parasites

Resistance to anti-malarial drugs is a widespread problem for control programmes for this devastating disease. Molecular tests are available for many anti-malarial drugs and are useful tools for the surveillance of drug resistance. However, the correlation of treatment outcome and molecular tests with particular parasite markers is not perfect, due in part to individuals who are able to clear genotypically drug-resistant parasites. This study aimed to identify molecular markers in the human genome that correlate with the clearance of malaria parasites after drug treatment, despite the drug resistance profile of the protozoan as predicted by molecular approaches

“Sexual” Population Structure and Genetics of the Malaria Agent P. falciparum

The population genetics and structure of P. falciparum determine the rate at which malaria evolves in response to interventions such as drugs and vaccines. This has been the source of considerable recent controversy, but here we demonstrate the organism to be essentially sexual, in an area of moderately high transmission in the Lower Shire Valley, Malawi. Seven thousand mosquitoes were collected and dissected, and genetic data were obtained on 190 oocysts from 56 infected midguts. The oocysts were genotyped at three microsatellite loci and the MSP1 locus. Selfing rate was estimated as 50% and there was significant genotypic linkage disequilibrium (LD) in the pooled oocysts. A more appropriate analysis searching for genotypic LD in outcrossed oocysts and/or haplotypic LD in the selfed oocysts found no evidence for LD, indicating that the population was effectively sexual. Inbreeding estimates at MSP1 were higher than at the microsatellites, possibly indicative of immune action against MSP1, but the effect was confounded by the probable presence of null mutations. Mating appeared to occur at random in mosquitoes and evidence regarding whether malaria clones in the same host were related (presumably through simultaneous inoculation in the same mosquito bite) was ambiguous. This is the most detailed genetic analysis yet of P. falciparum sexual stages, and shows P. falciparum to be a sexual organism whose genomes are in linkage equilibrium, which acts to slow the emergence of drug resistance and vaccine insensitivity, extending the likely useful therapeutic lifespan of drugs and vaccines

The biology of sexual development of Plasmodium: the design and implementation of transmission-blocking strategies

A meeting to discuss the latest developments in the biology of sexual development of Plasmodium and transmission-control was held April 5-6, 2011, in Bethesda, MD. The meeting was sponsored by the Bill & Melinda Gates Foundation and the National Institutes of Health, National Institute of Allergy and Infectious Diseases (NIH/NIAID) in response to the challenge issued at the Malaria Forum in October 2007 that the malaria community should re-engage with the objective of global eradication. The consequent rebalancing of research priorities has brought to the forefront of the research agenda the essential need to reduce parasite transmission. A key component of any transmission reduction strategy must be methods to attack the parasite as it passes from man to the mosquito (and vice versa). Such methods must be rationally based on a secure understanding of transmission from the molecular-, cellular-, population- to the evolutionary-levels. The meeting represented a first attempt to draw together scientists with expertise in these multiple layers of understanding to discuss the scientific foundations and resources that will be required to provide secure progress toward the design and successful implementation of effective interventions

The transmission potential of malaria-infected mosquitoes (An.gambiae-Keele, An.arabiensis-Ifakara) is altered by the vertebrate blood type they consume during parasite development

The efficiency of malaria parasite development within mosquito vectors (sporogony) is a critical determinant of transmission. Sporogony is thought to be controlled by environmental conditions and mosquito/parasite genetic factors, with minimal contribution from mosquito behaviour during the period of parasite development. We tested this assumption by investigating whether successful sporogony of Plasmodium falciparum parasites through to human-infectious transmission stages is influenced by the host species upon which infected mosquitoes feed. Studies were conducted on two major African vector species that generally are found to differ in their innate host preferences: Anopheles arabiensis and An. gambiae sensu stricto. We show that the proportion of vectors developing transmissible infections (sporozoites) was influenced by the source of host blood consumed during sporogony. The direction of this effect was associated with the innate host preference of vectors: higher sporozoite prevalences were generated in the usually human-specialist An. gambiae s.s. feeding on human compared to cow blood, whereas the more zoophilic An. arabiensis had significantly higher prevalences after feeding on cow blood. The potential epidemiological implications of these results are discussed