Dynamics of pfcrt alleles CVMNK and CVIET in chloroquine-treated Sudanese patients infected with Plasmodium falciparum

BACKGROUND: Parasite resistance to the anti-malarial drug chloroquine is common in eastern Sudan. Dynamic within-host changes in the relative abundance of both sensitive and resistant Plasmodium falciparum parasites were examined in a cohort of chloroquine-treated patients presenting with uncomplicated falciparum malaria, using a novel allele-specific quantitative approach. METHODS: Treatment outcomes were determined for 93 patients of all ages in a per protocol cohort using a modified 14-day WHO protocol. Parasite DNA samples at days 0, 1, 2, 3, 7 and 14 following treatment were analysed using real-time quantitative PCR methods that distinguished resistant and sensitive genotypes at amino acids 72-76 of the pfcrt locus. RESULTS: Chloroquine treatment was not efficacious, and of 93 assessable patients, only 10 individuals (10.7%; 95% C.I. 4.34-17.2%) enjoyed an adequate clinical and parasitological response. Resistant parasites with the haplotype CVIET at codons 72-76 of the pfcrt locus were dominant in the starting population. Chloroquine sensitive parasites with the haplotype CVMNK were detected in 19 individuals prior to treatment (20.43%; 95% C.I. 5.14-18.5%). In these patients, CQ treatment rapidly selected CVIET parasites, and this haplotype overwhelmingly dominated the parasite population in each individual by day 2 after treatment. CONCLUSIONS: Such rapid intra-host selection of particular genotypes after the introduction of drug will cause frequent misidentification of parasite genotypes present in the starting population. This will have a potentially serious confounding effect on clinical trials which employ PCR-corrected estimates of treatment failure, as resistant parasites below the detection threshold in the pre-treatment sample can be erroneously classified as "new" infections during follow-up, over-estimating drug efficacy

A comparative study on the efficacy of artesunate plus sulphadoxine/pyrimethamine versus artemether-lumefantrine in eastern Sudan

BACKGROUND: A combination of artesunate (AS) plus sulphadoxine/pyrimethamine (SP) as first-line and artemether-lumefantrine (AL) as second-line treatment are currently recommended against uncomplicated P. falciparum infection in Sudan. However, there is limited information on the efficacy of ACTs in the country and only one report of PCR-corrected results for AS/SP only. METHODS: The WHO protocol for the assessment of antimalarial drug efficacy for the treatment of uncomplicated falciparum malaria was employed. Artesunate plus sulphadoxine/pyrimethamine (AS/SP) was compared to artemether-lumefantrine (AL) in a 28-day follow up. Samples that were classified as early treatment failure (ETF), late treatment failure (LCF) or late parasitological failure (LPF) were genotyped for msp-1 and msp-2 genes to differentiate recrudescence from reinfection. RESULTS: A total of 178 patients were screened and 160 met the enrollment criteria and were recruited to the study of which 157 (98.1%) completed the follow up and had an analysed treatment outcome. On the AS/SP arm, three (0.038%) patients were lost during the follow-up, two on day 1 and one on day 7, and 77 (96.3) completed the study, while all 80 (100%) patients completed the follow up in the AL arm. In the per protocol analysis for AS/SP the treatment outcome for patients who completed the follow-up were as follows: adequate clinical and parasitological response (ACPR); 84.4% ETF; 1.3%, LCF; 3.9%, (LPF); 10.4%. For the AL arm the out come was as follows, ACPR; 90%, ETF; 0%, LCF; 6.3% and LPF; 3.8%. However, when PCR-corrected, 6.5% (5/77) of patients treated with AS/SP maintained parasites from their primary infection, while (7/80) in the AL group maintained their initial parasite genotype. Therefore, PCR-corrected efficacy was 93.5% in the AS/SP treated group and for AL it was 91.3%. CONCLUSION: Both AS/SP and AL are highly effective for the treatment of uncomplicated falciparum malaria in eastern Sudan. However, AS/SP appears to have a slightly higher efficacy than AL, this may be due to patient compliance with the repeated dose rather than drug efficacy

Spatial and temporal distribution of the malaria mosquito Anopheles arabiensis in northern Sudan: influence of environmental factors and implications for vector control

BACKGROUND: Malaria is an important public health problem in northern Sudan, but little is known about the dynamics of its transmission. Given the characteristic low densities of Anopheles arabiensis and the difficult terrain in this area, future vector control strategies are likely to be based on area-wide integrated pest management (AW-IPM) that may include the sterile insect technique (SIT). To support the planning and implementation of future AW-IPM activities, larval surveys were carried out to provide key data on spatial and seasonal dynamics of local vector populations. METHODS: Monthly cross-sectional larval surveys were carried out between March 2005 and May 2007 in two localities (Dongola and Merowe) adjacent to the river Nile. A stratified random sampling strategy based on the use of Remote Sensing (RS), Geographical Information Systems (GIS) and the Global Positioning System (GPS) was used to select survey locations. Breeding sites were mapped using GPS and data on larval density and breeding site characteristics were recorded using handheld computers. Bivariate and multivariate logistic regression models were used to identify breeding site characteristics associated with increased risk of presence of larvae. Seasonal patterns in the proportion of breeding sites positive for larvae were compared visually to contemporaneous data on climate and river height. RESULTS: Of a total of 3,349 aquatic habitats sampled, 321 (9.6%) contained An. arabiensis larvae. The frequency with which larvae were found varied markedly by habitat type. Although most positive sites were associated with temporary standing water around the margins of the main Nile channel, larvae were also found at brickworks and in areas of leaking pipes and canals - often far from the river. Close to the Nile channel, a distinct seasonal pattern in larval populations was evident and appeared to be linked to the rise and fall of the river level. These patterns were not evident in vector populations breeding in artificial water sources away from the river. CONCLUSION: The GIS-based survey strategy developed in this study provides key data on the population dynamics of An. arabiensis in Northern State. Quantitative estimates of the contributions of various habitat types and their proximity to settlements provide a basis for planning a strategy for reducing malaria risk by elimination of the vector population

Alternatively spliced transcripts and novel pseudogenes of the Plasmodium falciparum resistance-associated locus pfcrt detected in East African malaria patients.

OBJECTIVES: Polymorphisms in the lysosomal transporter encoded by the pfcrt gene directly impact on Plasmodium falciparum susceptibility to aminoquinolines. The Lys76Thr mutation is the critical change conferring chloroquine resistance in vitro and in vivo, but always occurs with additional non-synonymous changes in the pfcrt coding sequence. We sought to better describe pfcrt polymorphisms distal to codon 76. METHODS: Small-volume samples (≤ 500 μL) of parasite-infected blood collected directly from malaria patients presenting for treatment in Sudan and Tanzania were immediately preserved for RNA extraction. The pfcrt locus was amplified from cDNA preparations by nested PCR and sequenced directly to derive full-length mRNA sequences. RESULTS: In one of two sites in Sudan, two patients were found with an unorthodox spliced form of pfcrt mRNA in which two exons were skipped, but it was not possible to test for the presence of the putative protein products of these aberrant transcripts. Genomic DNA sequencing from dried blood spots collected in parallel confirmed the presence of spliced pfcrt pseudogenes in a minority of parasite isolates. Full-length cDNA from conventionally spliced mRNA molecules in all study sites demonstrated the existence of a variety of pfcrt haplotypes in East Africa, and thus provides evidence of intragenic recombination. CONCLUSIONS: The presence of pseudogenes, although unlikely to have any direct public health impact, may confound results obtained from simple genotyping methods that consider only codon 76 and the adjacent residues of pfcrt

Ethical, legal and social aspects of the approach in Sudan

The global malaria situation, especially in Africa, and the problems frequently encountered in chemical control of vectors such as insecticide resistance, emphasize the urgency of research, development and implementation of new vector control technologies that are applicable at regional and local levels. The successful application of the sterile insect technique (SIT) for the control of the New World screwworm Cochliomyia hominivorax and several species of fruit flies has given impetus to the use of this method for suppression or elimination of malaria vectors in some areas of Africa including Northern State of Sudan. The research and development phase of the Northern State feasibility study has been started. Sudanese stakeholders are working side-by-side with the International Atomic Energy Agency in the activities of this important phase. Several ethical, legal and social issues associated with this approach arose during this phase of the project. They need to be seriously considered and handled with care. In this paper, these issues are described, and the current and proposed activities to overcome potential hurdles to ensure success of the project are listed

Towards a sterile insect technique field release of Anopheles arabiensis mosquitoes in Sudan: Irradiation, transportation, and field cage experimentation

<p>Abstract</p> <p>Background</p> <p>The work described in this article forms part of a study to suppress a population of the malaria vector <it>Anopheles arabiensis </it>in Northern State, Sudan, with the Sterile Insect Technique. No data have previously been collected on the irradiation and transportation of anopheline mosquitoes in Africa, and the first series of attempts to do this in Sudan are reported here. In addition, experiments in a large field cage under near-natural conditions are described.</p> <p>Methods</p> <p>Mosquitoes were irradiated in Khartoum and transported as adults by air to the field site earmarked for future releases (400 km from the laboratory). The field cage was prepared for experiments by creating resting sites with favourable conditions. The mating and survival of (irradiated) laboratory males and field-collected males was studied in the field cage, and two small-scale competition experiments were performed.</p> <p>Results</p> <p>Minor problems were experienced with the irradiation of insects, mostly associated with the absence of a rearing facility in close proximity to the irradiation source. The small-scale transportation of adult mosquitoes to the release site resulted in minimal mortality (< 6%). Experiments in the field cage showed that mating occurred in high frequencies (i.e. an average of 60% insemination of females after one or two nights of mating), and laboratory reared males (i.e. sixty generations) were able to inseminate wild females at rates comparable to wild males. Based on wing length data, there was no size preference of males for mates. Survival of mosquitoes from the cage, based on recapture after mating, was satisfactory and approximately 60% of the insects were recaptured after one night. Only limited information on male competitiveness was obtained due to problems associated with individual egg laying of small numbers of wild females.</p> <p>Conclusion</p> <p>It is concluded that although conditions are challenging, there are no major obstacles associated with the small-scale irradiation and transportation of insects in the current setting. The field cage is suitable for experiments and studies to test the competitiveness of irradiated males can be pursued. The scaling up of procedures to accommodate much larger numbers of insects needed for a release is the next challenge and recommendations to further implementation of this genetic control strategy are presented.</p

Field site selection: getting it right first time around

The selection of suitable field sites for integrated control of Anopheles mosquitoes using the sterile insect technique (SIT) requires consideration of the full gamut of factors facing most proposed control strategies, but four criteria identify an ideal site: 1) a single malaria vector, 2) an unstructured, relatively low density target population, 3) isolation of the target population and 4) actual or potential malaria incidence. Such a site can exist in a diverse range of situations or can be created. Two contrasting SIT field sites are examined here: the desert-flanked Dongola Reach of the Nile River in Northern State, Sudan, where malaria is endemic, and the island of La Reunion, where autochthonous malaria is rare but risk is persistent. The single malaria-transmitting vector at both sites is Anopheles arabiensis. In Sudan, the target area is a narrow 500 km corridor stretching from the rocky terrain at the Fourth Cataract - just above the new Merowe Dam, to the northernmost edge of the species range, close to Egypt. Vector distribution and temporal changes in density depend on the Nile level, ambient temperature and human activities. On La Reunion, the An. arabiensis population is coastal, limited and divided into three areas by altitude and exposure to the trade winds on the east coast. Mosquito vectors for other diseases are an issue at both sites, but of primary importance on La Reunion due to the recent chikungunya epidemic. The similarities and differences between these two sites in terms of suitability are discussed in the context of area-wide integrated vector management incorporating the SIT

Polymorphisms in Plasmodium falciparum chloroquine resistance transporter and multidrug resistance 1 genes: parasite risk factors that affect treatment outcomes for P. falciparum malaria after artemether-lumefantrine and artesunate-amodiaquine.

Adequate clinical and parasitologic cure by artemisinin combination therapies relies on the artemisinin component and the partner drug. Polymorphisms in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes are associated with decreased sensitivity to amodiaquine and lumefantrine, but effects of these polymorphisms on therapeutic responses to artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) have not been clearly defined. Individual patient data from 31 clinical trials were harmonized and pooled by using standardized methods from the WorldWide Antimalarial Resistance Network. Data for more than 7,000 patients were analyzed to assess relationships between parasite polymorphisms in pfcrt and pfmdr1 and clinically relevant outcomes after treatment with AL or ASAQ. Presence of the pfmdr1 gene N86 (adjusted hazards ratio = 4.74, 95% confidence interval = 2.29 - 9.78, P < 0.001) and increased pfmdr1 copy number (adjusted hazards ratio = 6.52, 95% confidence interval = 2.36-17.97, P < 0.001 : were significant independent risk factors for recrudescence in patients treated with AL. AL and ASAQ exerted opposing selective effects on single-nucleotide polymorphisms in pfcrt and pfmdr1. Monitoring selection and responding to emerging signs of drug resistance are critical tools for preserving efficacy of artemisinin combination therapies; determination of the prevalence of at least pfcrt K76T and pfmdr1 N86Y should now be routine

A Randomized Open-Label Trial of Artesunate- Sulfadoxine-Pyrimethamine with or without Primaquine for Elimination of Sub-Microscopic P. falciparum Parasitaemia and Gametocyte Carriage in Eastern Sudan

In areas of seasonal malaria transmission, treatment of asymptomatic carriers of malaria parasites, whose parasitaemia persists at low densities throughout the dry season, could be a useful strategy for malaria control. We carried out a randomized trial to compare two drug regimens for clearance of parasitaemia in order to identify the optimum regimen for use in mass drug administration in the dry season.A two-arm open-label randomized controlled trial was conducted during the dry season in an area of distinct seasonal malaria in two villages in Gedarif State in eastern Sudan. Participants were asymptomatic adults and children aged over 6 months, with low-density P. falciparum infection detected by PCR. Participants were randomized to receive artesunate/sulfadoxine-pyrimethamine (AS+SP) combination for three days with or without a dose of primaquine (PQ) on the fourth day. Parasitaemia detected by PCR on days 3, 7 and 14 after the start of treatment and gametocytes detected by RT-PCR on days 7 and 14 were then recorded. 104 individuals who had low density parasitaemia at screening were randomized and treated during the dry season. On day 7, 8.3% were positive by PCR in the AS+SP+PQ group and 6.5% in the AS+SP group (risk difference 1.8%, 95%CI -10.3% to +13.8%). At enrolment, 12% (12/100) were carrying gametocytes. This was reduced to 6.4% and 4.4% by day 14 (Risk difference 1.9% (95%CI -9.3% to +13.2%) in AS+SP+PQ and AS+SP groups, respectively.Addition of primaquine to artemisinin combination treatment did not improve elimination of parasitaemia and prevention of gametocyte carriage in carriers with low-density parasitaemia in the dry season.ClinicalTrials.gov NCT00330902

Efficacy of Single-Dose Primaquine With Artemisinin Combination Therapy on Plasmodium falciparum Gametocytes and Transmission: An Individual Patient Meta-Analysis

Background Since the World Health Organization recommended single low-dose (0.25mg/kg) primaquine (PQ) in combination with artemisinin-based combination therapies (ACTs) in areas of low transmission or artemisinin-resistant P. falciparum, several single-site studies have been conducted to assess its efficacy. Methods An individual patient meta-analysis to assess the gametocytocidal and transmission-blocking efficacy of PQ used in combination with different ACTs was conducted. Random effects logistic regression was used to quantify PQ effect on (i) gametocyte carriage in the first two weeks post-treatment; (ii) the probability of infecting at least one mosquito or of a mosquito becoming infected. Results In 2,574 participants from fourteen studies, PQ reduced PCR-determined gametocyte carriage on days 7 and 14, most apparently in patients presenting with gametocytaemia on day 0 (Odds Ratio (OR)=0.22; 95%CI 0.17-0.28 and OR=0.12; 95%CI 0.08–0.16, respectively). The rate of decline in gametocyte carriage was faster when PQ was combined with artemether-lumefantrine (AL) compared to dihydroartemisinin-piperaquine (DP) (p=0.010 for day 7). Addition of 0.25mg/kg PQ was associated with near complete prevention of transmission to mosquitoes. Conclusion Primaquine’s transmission-blocking effects are achieved with 0.25 mg/kg PQ. Gametocyte persistence and infectivity are lower when PQ is combined with AL compared to DP