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 (0.25 mg/kg) primaquine (PQ) in combination with artemisinin-based combination therapies (ACTs) in areas of low transmission or artemisinin-resistant Plasmodium falciparum, several single-site studies have been conducted to assess efficacy. METHODS: An individual patient meta-analysis to assess gametocytocidal and transmission-blocking efficacy of PQ in combination with different ACTs was conducted. Random effects logistic regression was used to quantify PQ effect on (1) gametocyte carriage in the first 2 weeks post treatment; and (2) the probability of infecting at least 1 mosquito or of a mosquito becoming infected. RESULTS: In 2574 participants from 14 studies, PQ reduced PCR-determined gametocyte carriage on days 7 and 14, most apparently in patients presenting with gametocytemia on day 0 (odds ratio [OR], 0.22; 95% confidence interval [CI], .17-.28 and OR, 0.12; 95% CI, .08-.16, respectively). Rate of decline in gametocyte carriage was faster when PQ was combined with artemether-lumefantrine (AL) compared to dihydroartemisinin-piperaquine (DP) (P = .010 for day 7). Addition of 0.25 mg/kg PQ was associated with near complete prevention of transmission to mosquitoes. CONCLUSIONS: Transmission blocking is achieved with 0.25 mg/kg PQ. Gametocyte persistence and infectivity are lower when PQ is combined with AL compared to DP.

Epidemiology and distribution of Plasmodium vivax malaria in Sudan.

BACKGROUND: Plasmodium vivax malaria has been recognised as an important cause of morbidity in several African countries. The prevalence was previously estimated as 2-5% in eastern Sudan. These estimates are observed to be rising and spreading continuously. The present study was undertaken to investigate the situation of distribution and epidemiology of P. vivax malaria in Sudan. METHODS: Cross-sectional malaria surveys carried out in hospitals and health centres covered 21 sites in 10 states. Data and blood samples were collected from 1226 clinically investigated suspected malaria cases of both genders and all ages. Microscopically detected malaria parasites were confirmed by PCR. RESULTS: The overall prevalence of P. vivax among the malaria cases was 26.6%. The prevalence showed significant variations between the states (p<0.001), which could be explained by differences in population movement, the presence of refugees and proximity to endemic neighbouring countries. It also varied significantly with residence status (p<0.001), reflecting the stability of transmission. CONCLUSION: Although malaria in Sudan is still largely attributed to Plasmodium falciparum, P. vivax has been rising with worrying proportions and spreading to new areas. The emergence and marked increase of P. vivax poses new challenges to malaria treatment and control in Sudan.

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.

Participation of irradiated Anopheles arabiensis males in swarms following field release in Sudan.

BACKGROUND: The success of the sterile insect technique (SIT) depends the release of large numbers of sterile males, which are able to compete for mates with the wild male population within the target area. Unfortunately, the processes of colonisation, mass production and irradiation may reduce the competitiveness of sterile males through genetic selection, loss of natural traits and somatic damage. In this context, the capacity of released sterile Anopheles arabiensis males to survive, disperse and participate in swarms at occurring at varying distances from the release site was studied using mark-release-recapture (MRR) techniques. METHODS: In order to assess their participation in swarms, irradiated and marked laboratory-reared male mosquitoes were released 50, 100 or 200 m from the known site of a large swarm on three consecutive nights. Males were collected from this large swarm on subsequent nights. Over the three days a total of 8,100 males were released. Mean distance travelled (MDT), daily probability of survival and estimated population size were calculated from the recapture data. An effect of male age at the time of release on these parameters was observed. RESULTS: Five per cent of the males released over three days were recaptured. In two-, three- and four-day-old males, MDT was 118, 178 and 170 m, and the daily survival probability 0.95, 0.90 and 0.75, respectively. From the recapture data on the first day following each release, the Lincoln index gives an estimation of 32,546 males in the natural population. DISCUSSION: Sterile An. arabiensis males released into the field were able to find and participate in existing swarms, and possibly even initiate swarms. The survival probability decreased with the age of male on release but the swarm participation and the distance travelled by older males seemed higher than for younger males. The inclusion of a pre-release period may thus be beneficial to male competitiveness and increase the attractiveness of adult sexing techniques, such as blood spiking.

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.

Swarming and mating behavior of male Anopheles arabiensis Patton (Diptera: Culicidae) in an area of the Sterile Insect Technique Project in Dongola, northern Sudan.

The problems facing the conventional mosquito control methods including resistance to insecticides have led to the development of alternative methods such as the Sterile Insect Technique (SIT) to suppress populations of the malaria vector Anopheles arabiensis in northern Sudan. This method entails the release of large numbers of irradiated males to compete against wild conspecifics for mating with virgin females in the field. The swarming and mating behaviors of this species were conducted at two field sites during the period 2009-2012 in Dongola, northern Sudan. Observations were made in the field sites and in a contained semi-field enclosure. In addition, participation of released irradiated-marked males in the swarms of wild mosquito was investigated. Swarms were observed on sunset in the vicinity of larval habitats around irrigation channel and stopped with the onset of the darkness about 21-25 min after the start. Swarms were observed above visual markers such as palm trees, bare ground, and manure. Several couples were observed leaving the swarms in copula in the direction of the sunlight. The majority of copulations were observed within 12-15 min of the start of swarming. Relatively low insemination rates (28%) of females collected from coupling pairs were observed. Irradiated-marked males were observed to join the natural swarms regularly, indicating their probable competitiveness with the other wild males. These findings enhance the feasibility of staging an SIT campaign against malaria vector in Northern State-Sudan.

Submicroscopic Plasmodium falciparum malaria and low birth weight in an area of unstable malaria transmission in Central Sudan.

BACKGROUND: Malaria, which frequently occurs in pregnant women in the tropics, is a leading cause of maternal anaemia and low birth weight (LBW) in infants. Few data exist concerning malaria infections that are present at submicroscopic levels during pregnancy and their LBW delivery in babies. METHODS: A case-control study (87 in each group) was conducted at the Medani Hospital, Central Sudan. Cases were women who had LBW deliveries where the infants weighed < 2,500 g. Controls were parturient women without having LBW babies. Obstetrical and medical characteristics were gathered from both groups through structured questionnaires. Both cases and controls were investigated for malaria using microscopic blood film analysis, placental histology and polymerase chain reaction (PCR). Microscopic and PCR analyses were conducted on maternal peripheral blood, placenta, and umbilical cord samples. Infant weights were recorded immediately after birth. RESULTS: Plasmodium falciparum-positive blood films were not obtained from any of the women (cases or controls). Twenty-seven (31.0%) versus 22 (25.3%) (P = 0.500) of the cases and controls, respectively, had placental malaria infections as determined by histological examination. In comparison to the controls, the submicroscopic malaria infection prevalence rates were significantly higher in the cases; 24 (27.6%) vs six (7.0%), P < 0.001. Multivariate analysis showed that while malaria infection of the placenta (based on histology) was not associated with LBW, submicroscopic P. falciparum infection (OR = 6.89, 95% CI = 2.2-20.8; P = 0.001), or a combination of histologically determined and submicroscopic infections (OR = 2.45, 95% CI = 1.2-4.9; P = 0.012), were significantly associated with LBW. CONCLUSION: In Central Sudan, pregnant women were at a higher risk of having an LBW delivery if they had submicroscopic infections rather than a histological diagnosis of placental malaria.

Increased pfmdr1 copy number and sequence polymorphisms in Plasmodium falciparum isolates from Sudanese malaria patients treated with artemether-lumefantrine.

Molecular markers for surveillance of Plasmodium falciparum resistance to current antimalarials are sorely needed. A 28-day efficacy study of artemether-lumefantrine in eastern Sudan identified 5 treatment failures among 100 evaluable patients; 9 further individuals were parasite positive by PCR during follow-up. Polymorphisms in pfatpase6 and pfmdr1 were evaluated by DNA sequencing. One individual carried parasites with a novel pfmdr1 polymorphism (F1044L). pfmdr1 gene amplification in parasites prior to treatment occurred in three individuals who had recurrent infection during follow-up.

Prolonged colonisation, irradiation, and transportation do not impede mating vigour and competitiveness of male Anopheles arabiensis mosquitoes under semi-field conditions in Northern Sudan.

Background: In Sudan, the Sterile Insect Technique (SIT) is being developed to suppress populations of Anopheles arabiensis. The present study was carried out to evaluate the impact of long-term colonisation, irradiation, and transportation on male vigour and mating competitiveness under controlled semi-field conditions. Materials and Methods: Male mosquitoes were irradiated in Khartoum as pupae and transported 400 km to the field site in Dongola. Wild males and females were collected as immature stages (larvae and pupae) from the field site and sexed immediately after adult emergence. Competition experiments were carried out to test the mating competitiveness and vigour of colonised males (non-irradiated or irradiated) against wild conspecifics in the semi-field system. Results: Mortality resulting from packaging and transportation from Khartoum to Dongola was low for adults (1.1% for irradiated and 1.3% for non-irradiated males). In contrast, all irradiated pupae died on their way to the field site. On average, 54.9% females were inseminated after one night. There were no differences between the number of females inseminated by colony males and those inseminated by wild males. Only a slightly significant difference between the numbers of females inseminated by irradiated males (14.0±1.7) or by wild males (19.7±1.7) was observed. However, the competitive index (CI) for irradiated and colony males when competed with wild males were 0.71 and 0.81 respectively. Conclusions: Packing and transportation methods for pupae need to be improved. Prolonged colonisation (68 generations), irradiation and transportation of adult males did not affect their ability to locate virgin females and compete against wild conspecifics. Irradiation, in contrast to many reports, only had a marginal effect on released males during the first night after their release. These findings support the feasibility of staging an SIT campaign against this malaria vector.

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.

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.

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.