Teun Bousema

Epidemiology and Infectivity of Plasmodium falciparum and Plasmodium vivax Gametocytes in Relation to Malaria Control and Elimination

SUMMARY Malaria remains a major cause of morbidity and mortality in the tropics, with Plasmodium falciparum responsible for the majority of the disease burden and P. vivax being the geographically most widely distributed cause of malaria. Gametocytes are the sexual-stage parasites that infect Anopheles mosquitoes and mediate the onward transmission of the disease. Gametocytes are poorly studied despite this crucial role, but with a recent resurgence of interest in malaria elimination, the study of gametocytes is in vogue. This review highlights the current state of knowledge with regard to the development and longevity of P. falciparum and P. vivax gametocytes in the human host and the factors influencing their distribution within endemic populations. The evidence for immune responses, antimalarial drugs, and drug resistance influencing infectiousness to mosquitoes is reviewed. We discuss how the application of molecular techniques has led to the identification of submicroscopic gametocyte carriage and to a reassessment of the human infectious reservoir. These components are drawn together to show how control measures that aim to reduce malaria transmission, such as mass drug administration and a transmission-blocking vaccine, might better be deployed.

Factors determining the occurrence of submicroscopic malaria infections and their relevance for control

Malaria parasite prevalence in endemic populations is an essential indicator for monitoring the progress of malaria control, and has traditionally been assessed by microscopy. However, surveys increasingly use sensitive molecular methods that detect higher numbers of infected individuals, questioning our understanding of the true infection burden and resources required to reduce it. Here we analyse a series of data sets to characterize the distribution and epidemiological factors associated with low-density, submicroscopic infections. We show that submicroscopic parasite carriage is common in adults, in low-endemic settings and in chronic infections. We find a strong, non-linear relationship between microscopy and PCR prevalence in population surveys (n=106), and provide a tool to relate these measures. When transmission reaches very low levels, submicroscopic carriers are estimated to be the source of 20–50% of all human-to-mosquito transmissions. Our findings challenge the idea that individuals with little previous malaria exposure have insufficient immunity to control parasitaemia and suggest a role for molecular screening.

Hitting hotspots: spatial targeting of malaria for control and elimination.

Teun Bousema and colleagues argue that targeting malaria “hotspots” is a highly efficient way to reduce malaria transmission at all levels of transmission intensity.

Asymptomatic malaria infections: detectability, transmissibility and public health relevance

Most Plasmodium falciparum infections that are detected in community surveys are characterized by low-density parasitaemia and the absence of clinical symptoms. Molecular diagnostics have shown that this asymptomatic parasitic reservoir is more widespread than previously thought, even in low-endemic areas. In this Opinion article, we describe the detectability of asymptomatic malaria infections and the relevance of submicroscopic infections for parasite transmission to mosquitoes and for community interventions that aim at reducing transmission. We argue that wider deployment of molecular diagnostic tools is needed to provide adequate insight into the epidemiology of malaria and infection dynamics to aid elimination efforts.

Identification of hot spots of malaria transmission for targeted malaria control.

BACKGROUND Variation in the risk of malaria within populations is a frequently described but poorly understood phenomenon. This heterogeneity creates opportunities for targeted interventions but only if hot spots of malaria transmission can be easily identified. METHODS We determined spatial patterns in malaria transmission in a district in northeastern Tanzania, using malaria incidence data from a cohort study involving infants and household-level mosquito sampling data. The parasite prevalence rates and age-specific seroconversion rates (SCRs) of antibodies against Plasmodium falciparum antigens were determined in samples obtained from people attending health care facilities. RESULTS Five clusters of higher malaria incidence were detected and interpreted as hot spots of transmission. These hot spots partially overlapped with clusters of higher mosquito exposure but could not be satisfactorily predicted by a probability model based on environmental factors. Small-scale local variation in malaria exposure was detected by parasite prevalence rates and SCR estimates for samples of health care facility attendees. SCR estimates were strongly associated with local malaria incidence rates and predicted hot spots of malaria transmission with 95% sensitivity and 85% specificity. CONCLUSIONS Serological markers were able to detect spatial variation in malaria transmission at the microepidemiological level, and they have the potential to form an effective method for spatial targeting of malaria control efforts.

Revisiting the circulation time of Plasmodium falciparum gametocytes: molecular detection methods to estimate the duration of gametocyte carriage and the effect of gametocytocidal drugs.

BackgroundThere is renewed acknowledgement that targeting gametocytes is essential for malaria control and elimination efforts. Simple mathematical models were fitted to data from clinical trials in order to determine the mean gametocyte circulation time and duration of gametocyte carriage in treated malaria patients.MethodsData were used from clinical trials from East Africa. The first trial compared non-artemisinin combination therapy (non-ACT: sulphadoxine-pyrimethamine (SP) plus amodiaquine) and artemisinin-based combination therapy (ACT: SP plus artesunate (AS) or artemether-lumefantrine). The second trial compared ACT (SP+AS) with ACT in combination with a single dose of primaquine (ACT-PQ: SP+AS+PQ). Mature gametocytes were quantified in peripheral blood samples by nucleic acid sequence based amplification. A simple deterministic compartmental model was fitted to gametocyte densities to estimate the circulation time per gametocyte; a similar model was fitted to gametocyte prevalences to estimate the duration of gametocyte carriage after efficacious treatment.ResultsThe mean circulation time of gametocytes was 4.6-6.5 days. After non-ACT treatment, patients were estimated to carry gametocytes for an average of 55 days (95% CI 28.7 - 107.7). ACT reduced the duration of gametocyte carriage fourfold to 13.4 days (95% CI 10.2-17.5). Addition of PQ to ACT resulted in a further fourfold reduction of the duration of gametocyte carriage.ConclusionsThese findings confirm previous estimates of the circulation time of gametocytes, but indicate a much longer duration of (low density) gametocyte carriage after apparently successful clearance of asexual parasites. ACT shortened the period of gametocyte carriage considerably, and had the most pronounced effect on mature gametocytes when combined with PQ.

Dried blood spots as a source of anti-malarial antibodies for epidemiological studies

BackgroundBlood spots collected onto filter paper are an established and convenient source of antibodies for serological diagnosis and epidemiological surveys. Although recommendations for the storage and analysis of small molecule analytes in blood spots exist, there are no published systematic studies of the stability of antibodies under different storage conditions.MethodsBlood spots, on filter paper or glass fibre mats and containing malaria-endemic plasma, were desiccated and stored at various temperatures for different times. Eluates of these spots were assayed for antibodies against two Plasmodium falciparum antigens, MSP-119 and MSP2, and calculated titres used to fit an exponential (first order kinetic) decay model. The first order rate constants (k) for each spot storage temperature were used to fit an Arrhenius equation, in order to estimate the thermal and temporal stability of antibodies in dried blood spots. The utility of blood spots for serological assays was confirmed by comparing antibodies eluted from blood spots with the equivalent plasma values in a series of samples from North Eastern Tanzania and by using blood spot-derived antibodies to estimate malaria transmission intensity in this site and for two localities in Uganda.ResultsAntibodies in spots on filter paper and glass fibre paper had similar stabilities but blood was more easily absorbed onto filter papers than glass fibre, spots were more regular and spot size was more closely correlated with blood volume for filter paper spots. Desiccated spots could be stored at or below 4°C for extended periods, but were stable for only very limited periods at ambient temperature. When desiccated, recoveries of antibodies that are predominantly of IgG1 or IgG3 subclasses were similar. Recoveries of antibodies from paired samples of serum and of blood spots from Tanzania which had been suitably stored showed similar recoveries of antibodies, but spots which had been stored for extended periods at ambient humidity and temperature showed severe loss of recoveries. Estimates of malaria transmission intensity obtained from serum and from blood spots were similar, and values obtained using blood spots agreed well with entomologically determined values.ConclusionThis study has demonstrated the suitability of filter paper blood spots paper for collection of serum antibodies, and provided clear guidelines for the treatment and storage of filter papers which emphasize the importance of desiccation and minimisation of time spent at ambient temperatures. A recommended protocol for collecting, storing and assaying blood spots is provided.

Substantial Contribution of Submicroscopical Plasmodium falciparum Gametocyte Carriage to the Infectious Reservoir in an Area of Seasonal Transmission

Background Man to mosquito transmission of malaria depends on the presence of the sexual stage parasites, gametocytes, that often circulate at low densities. Gametocyte densities below the microscopical threshold of detection may be sufficient to infect mosquitoes but the importance of submicroscopical gametocyte carriage in different transmission settings is unknown. Methodology/Principal Findings Membrane feeding experiments were carried out on 80 children below 14 years of age at the end of the wet season in an area of seasonal malaria transmission in Burkina Faso. Gametocytes were quantified by microscopy and by Pfs25-based quantitative nucleic acid sequence-based amplification assay (QT-NASBA). The childrens infectiousness was determined by membrane feeding experiments in which a venous blood sample was offered to locally reared Anopheles mosquitoes. Gametocytes were detected in 30.0% (24/80) of the children by microscopy compared to 91.6% (65/71) by QT-NASBA (p<0.001). We observed a strong association between QT-NASBA gametocyte density and infection rates (p = 0.007). Children with microscopically detectable gametocytes were more likely to be infectious (68.2% compared to 31.7% of carriers of submicroscopical gametocytes, p = 0.001), and on average infected more mosquitoes (13.2% compared to 2.3%, p<0.001). However, because of the high prevalence of submicroscopical gametocyte carriage in the study population, carriers of sub-microscopical gametocytes were responsible for 24.2% of the malaria transmission in this population. Conclusions/Significance Submicroscopical gametocyte carriage is common in an area of seasonal transmission in Burkina Faso and contributes substantially to the human infectious reservoir. Submicroscopical gametocyte carriage should therefore be considered when implementing interventions that aim to reduce malaria transmission.

Reduction of transmission from malaria patients by artemisinin combination therapies: a pooled analysis of six randomized trials.

BackgroundArtemisinin combination therapies (ACT), which are increasingly being introduced for treatment of Plasmodium falciparum malaria, are more effective against sexual stage parasites (gametocytes) than previous first-line antimalarials and therefore have the potential to reduce parasite transmission. The size of this effect is estimated in symptomatic P. falciparum infections.MethodsData on 3,174 patients were pooled from six antimalarial trials conducted in The Gambia and Kenya. Multivariable regression was used to investigate the role of ACT versus non-artemisinin antimalarial treatment, treatment failure, presence of pre-treatment gametocytes and submicroscopic gametocytaemia on transmission to mosquitoes and the area under the curve (AUC) of gametocyte density during the 28 days of follow up.ResultsACT treatment was associated with a significant reduction in the probability of being gametocytaemic on the day of transmission experiments (OR 0.20 95% CI 0.16–0.26), transmission to mosquitoes by slide-positive gametocyte carriers (OR mosquito infection 0.49 95% CI 0.33–0.73) and AUC of gametocyte density (ratio of means 0.35 95% CI 0.31–0.41). Parasitological treatment failure did not account for the difference between ACT and non-artemisinin impact. The presence of slide-positive gametocytaemia prior to treatment significantly reduced ACT impact on gametocytaemia (p < 0.001). Taking account of submicroscopic gametocytaemia reduced estimates of ACT impact in a high transmission setting in Kenya, but not in a lower transmission setting in the Gambia.ConclusionTreatment with ACT significantly reduces infectiousness of individual patients with uncomplicated falciparum malaria compared to previous first line treatments. Rapid treatment of cases before gametocytaemia is well developed may enhance the impact of ACT on transmission.

Primaquine Clears Submicroscopic Plasmodium falciparum Gametocytes that Persist after Treatment with Sulphadoxine-Pyrimethamine and Artesunate

Background P. falciparum gametocytes may persist after treatment with sulphadoxine-pyrimethamine (SP) plus artesunate (AS) and contribute considerably to malaria transmission. We determined the efficacy of SP+AS plus a single dose of primaquine (PQ, 0.75 mg/kg) on clearing gametocytaemia measured by molecular methods. Methodology The study was conducted in Mnyuzi, an area of hyperendemic malaria in north-eastern Tanzania. Children aged 3–15 years with uncomplicated P. falciparum malaria with an asexual parasite density between 500–100,000 parasites/µL were randomized to receive treatment with either SP+AS or SP+AS+PQ. P. falciparum gametocyte prevalence and density during the 42-day follow-up period were determined by real-time nucleic acid sequence-based amplification (QT-NASBA). Haemoglobin levels (Hb) were determined to address concerns about haemolysis in G6PD-deficient individuals. Results 108 individuals were randomized. Pfs25 QT-NASBA gametocyte prevalence was 88–91% at enrolment and decreased afterwards for both treatment arms. Gametocyte prevalence and density were significantly lower in children treated with SP+AS+PQ. On day 14 after treatment 3.9% (2/51) of the SP+AS+PQ treated children harboured gametocytes compared to 62.7% (32/51) of those treated with SP+AS (p<0.001). Hb levels were reduced in the week following treatment with SP+AS+PQ and this reduction was related to G6PD deficiency. The Hb levels of all patients recovered to pre-treatment levels or greater within one month after treatment. Conclusions PQ clears submicroscopic gametocytes after treatment with SP+AS and the persisting gametocytes circulated at densities that are unlikely to contribute to malaria transmission. For individuals without severe anaemia, addition of a single dose of PQ to an efficacious antimalarial drug combination is a safe approach to reduce malaria transmission following treatment. Trial Registration Controlled-Trials.com ISRCTN61534963