Increased Plasmodium falciparum gametocyte production in mixed infections with P. malariae.

Plasmodium falciparum and P. malariae occur endemically in many parts of Africa. Observations from malariotherapy patients suggest that co-infection with P. malariae may increase P. falciparum gametocyte production. We determined P. falciparum gametocyte prevalence and density by quantitative nucleic acid sequence-based amplification (QT-NASBA) after antimalarial treatment of Kenyan children with either P. falciparum mono-infection or P. falciparum and P. malariae mixed infection. In addition, we analyzed the relationship between mixed species infections and microscopic P. falciparum gametocyte prevalence in three datasets from previously published studies. In Kenyan children, QT-NASBA gametocyte density was increased in mixed species infections (P = 0.03). We also observed higher microscopic prevalences of P. falciparum gametocytes in mixed species infections in studies from Tanzania and Kenya (odds ratio = 2.15, 95% confidence interval = 0.99-4.65 and 2.39, 1.58-3.63) but not in a study from Nigeria. These data suggest that co-infection with P. malariae is correlated with increased P. falciparum gametocytemia.

Submicroscopic Plasmodium falciparum gametocyte densities frequently result in mosquito infection.

Submicroscopic Plasmodium falciparum gametocytemia (<5,000 gametocytes/mL) is common and may result in mosquito infection. We assessed the relation between gametocyte density and mosquito infection under experimental and field conditions using real-time quantitative nucleic acid sequence-based amplification (QT-NASBA) for gametocyte quantification. Serial dilutions of NF54 P. falciparum gametocytes showed a positive association between gametocyte density and the proportion of infected mosquitoes (beta=6.1; 95% confidence interval [CI], 2.7-9.6; P=0.001). Successful infection became unlikely below an estimated density of 250-300 gametocytes/mL. In the field, blood samples of 100 naturally infected children showed a positive association between gametocyte density and oocyst counts in mosquitoes (beta=0.38; 95% CI, 0.14-0.61; P=0.002). The relative contribution to malaria transmission was similar for carriers with submicroscopic and microscopic gametocytemia. Our results show that transmission occurs efficiently at submicroscopic gametocyte densities and that carriers harboring submicroscopic gametocytemia constitute a considerable proportion of the human infectious reservoir.

Larval habitats of Anopheles gambiae s.s. (Diptera: Culicidae) influences vector competence to Plasmodium falciparum parasites.

BACKGROUND: The origin of highly competent malaria vectors has been linked to productive larval habitats in the field, but there isn't solid quantitative or qualitative data to support it. To test this, the effect of larval habitat soil substrates on larval development time, pupation rates and vector competence of Anopheles gambiae to Plasmodium falciparum were examined. METHODS: Soils were collected from active larval habitats with sandy and clay substrates from field sites and their total organic matter estimated. An. gambiae larvae were reared on these soil substrates and the larval development time and pupation rates monitored. The emerging adult mosquitoes were then artificially fed blood with infectious P. falciparum gametocytes from human volunteers and their midguts examined for oocyst infection after seven days. The wing sizes of the mosquitoes were also measured. The effect of autoclaving the soil substrates was also evaluated. RESULTS: The total organic matter was significantly different between clay and sandy soils after autoclaving (P = 0.022). A generalized liner model (GLM) analysis identified habitat type (clay soil, sandy soil, or lake water) and autoclaving (that reduces presence of microbes) as significant factors affecting larval development time and oocyst infection intensities in adults. Autoclaving the soils resulted in the production of significantly smaller sized mosquitoes (P = 0.008). Autoclaving clay soils resulted in a significant reduction in Plasmodium falciparum oocyst intensities (P = 0.041) in clay soils (unautoclaved clay soils (4.28 +/- 0.18 oocysts/midgut; autoclaved clay soils = 1.17 +/- 0.55 oocysts/midgut) although no difference (P = 0.480) in infection rates was observed between clay soils (10.4%), sandy soils (5.3%) or lake water (7.9%). CONCLUSION: This study suggests an important nutritional role for organic matter and microbial fauna on mosquito fitness and vector competence. It shows that the quality of natural aquatic habitats of mosquito larvae may influence malaria parasite transmission potential by An. gambiae. This information can be important in targeting larval habitats for malaria control.

Effect of discriminative plant-sugar feeding on the survival and fecundity of Anopheles gambiae.

BACKGROUND: A previous study showed for Anopheles gambiae s.s. a gradation of feeding preference on common plant species growing in a malaria holoendemic area in western Kenya. The present follow-up study determines whether there is a relationship between the mosquito's preferences and its survival and fecundity. METHODS: Groups of mosquitoes were separately given ad libitum opportunity to feed on five of the more preferred plant species (Hamelia patens, Parthenium hysterophorus, Ricinus communis, Senna didymobotrya, and Tecoma stans) and one of the less preferred species (Lantana camara). The mosquitoes were monitored daily for survival. Sugar solution (glucose 6%) and water were used as controls. In addition, the fecundity of mosquitoes on each plant after (i) only one blood meal (number of eggs oviposited), and (ii) after three consecutive blood meals (proportion of females ovipositing, number of eggs oviposited and hatchability of eggs), was determined. The composition and concentration of sugar in the fed-on parts of each plant species were determined using gas chromatography. Using SAS statistical package, tests for significant difference of the fitness values between mosquitoes exposed to different plant species were conducted. RESULTS AND CONCLUSION: Anopheles gambiae that had fed on four of the five more preferred plant species (T. stans, S. didymobotrya, R. communis and H. patens, but not P. hysterophorus) lived longer and laid more eggs after one blood meal, when compared with An. gambiae that had fed on the least preferred plant species L. camara. When given three consecutive blood-meals, the percentage of females that oviposited, but not the number of eggs laid, was significantly higher for mosquitoes that had previously fed on the four more preferred plant species. Total sugar concentration in the preferred plant parts was significantly correlated with survival and with the proportion of females that laid eggs. This effect was associated mainly with three sugar types, namely glucose, fructose, and gulose. Except for P. hysterophorus, the results suggest that feeding by mosquitoes on preferred plant species under natural conditions results in higher fitness-related benefits, and that the sugar content in preferred plant parts is largely responsible for these effects.

The presence of Plasmodium falciparum gametocytes in human blood increases the gravidity of Anopheles gambiae mosquitoes.

We conducted a field study in an area of endemic malaria transmission in western Kenya to determine whether mosquitoes that feed on gametocyte-infected blood but do not become infected have reduced or enhanced fecundity in comparison to mosquitoes fed on uninfected blood. Fifteen paired membrane-feeding experiments were conducted in which two strains of Anopheles gambiae mosquitoes were simultaneously fed on either Plasmodium falciparum-infected blood from children or uninfected control blood from adults. The presence of noninfecting gametocytes in blood increased the probability that An. gambiae would produce eggs after one blood meal by sixfold (odds ratio for control relative to infected blood group 0.16; 95% CI 0.10-0.23). This result could not be explained by variation in blood meal size or hemoglobin content between hosts. When children cleared their infections, the difference in gravidity between mosquitoes fed on their blood and uninfected adults disappeared, suggesting this phenomenon is due to the presence of Plasmodium gametocytes in blood and not to host-specific factors such as age. This result was observed in two mosquito strains that differ in their innate fecundity, suggesting it may apply generally. To our knowledge, this is the first time that Plasmodium has been implicated as enhancing vector gravidity.

Host genotype by parasite genotype interactions underlying the resistance of anopheline mosquitoes to Plasmodium falciparum.

BACKGROUND: Most studies on the resistance of mosquitoes to their malaria parasites focus on the response of a mosquito line or colony against a single parasite genotype. In natural situations, however, it may be expected that mosquito-malaria relationships are based, as are many other host-parasite systems, on host genotype by parasite genotype interactions. In such systems, certain hosts are resistant to one subset of the parasite's genotypes, while other hosts are resistant to a different subset. METHODS: To test for genotype by genotype interactions between malaria parasites and their anopheline vectors, different genetic backgrounds (families consisting of the F1 offspring of individual females) of the major African vector Anopheles gambiae were challenged with several isolates of the human malaria parasite Plasmodium falciparum (obtained from naturally infected children in Kenya). RESULTS: Averaged across all parasites, the proportion of infected mosquitoes and the number of oocysts found in their midguts were similar in all mosquito families. Both indices of resistance, however, differed considerably among isolates of the parasite. In particular, no mosquito family was most resistant to all parasites, and no parasite isolate was most infectious to all mosquitoes. CONCLUSIONS: These results suggest that the level of mosquito resistance depends on the interaction between its own and the parasite's genotype. This finding thus emphasizes the need to take into account the range of genetic diversity exhibited by mosquito and malaria field populations in ideas and studies concerning the control of malaria.

Eradication of Anopheles gambiae from Brazil: lessons for malaria control in Africa?

Current malaria-control strategies emphasise domestic protection against adult mosquitoes with insecticides, and improved access to medical services. Malaria prevention by killing adult mosquitoes is generally favoured because moderately reducing their longevity can radically suppress community-level transmission. By comparison, controlling larvae has a less dramatic effect at any given level of coverage and is often more difficult to implement. Nevertheless, the historically most effective campaign against African vectors is the eradication of accidentally introduced Anopheles gambiae from 54000 km(2) of largely ideal habitat in northeast Brazil in the 1930s and early 1940s. This outstanding success was achieved through an integrated programme but relied overwhelmingly upon larval control. This experience was soon repeated in Egypt and another larval control programme successfully suppressed malaria for over 20 years around a Zambian copper mine. These affordable approaches were neglected after the advent of dichlorodiphenyl trichloroethane (DDT) and global malaria-control policy shifted toward domestic adulticide methods. Larval-control methods should now be re-prioritised for research, development, and implementation as an additional way to roll back malaria.

Plasmodium falciparum gametocyte carriage in asymptomatic children in western Kenya.

BACKGROUND: Studies on Plasmodium falciparum gametocyte development and dynamics have almost exclusively focused on patients treated with antimalarial drugs, while the majority of parasite carriers in endemic areas are asymptomatic. This study identified factors that influence gametocytaemia in asymptomatic children in the absence and presence of pyrimethamine-sulphadoxine (SP) antimalarial treatment. METHODS: A cohort of 526 children (6 months-16 years) from western Kenya was screened for asexual parasites and gametocytes and followed weekly up to four weeks. Children with an estimated parasitaemia of > or =1,000 parasites/microl were treated with SP according to national guidelines. Factors associated with gametocyte development and persistence were determined in untreated and SP-treated children with P. falciparum mono-infection. RESULTS: Gametocyte prevalence at enrollment was 33.8% in children below five years of age and decreased with age. In the absence of treatment 18.6% of the children developed gametocytaemia during follow-up; in SP-treated children this proportion was 29.8%. Age, high asexual parasite density and gametocyte presence at enrollment were predictive factors for gametocytaemia. The estimated mean duration of gametocytaemia for children below five, children from five to nine and children ten years and above was 9.4, 7.8 and 4.1 days, respectively. CONCLUSION: This study shows that a large proportion of asymptomatic untreated children develop gametocytaemia. Gametocytaemia was particularly common in children below five years who harbor gametocytes for a longer period of time. The age-dependent duration of gametocytaemia has not been previously shown and could increase the importance of this age group for the infectious reservoir.

The development of Plasmodium falciparum in experimentally infected Anopheles gambiae (Diptera: Culicidae) under ambient microhabitat temperature in western Kenya.

The effect of microhabitat temperature variation on the early development of Plasmodium falciparum in experimentally infected Anopheles gambiae s.s. (Diptera: Culicidae) was studied. Batches of mosquitoes were fed artificially on gametocyteamic blood obtained from human volunteers and then held in five environmental conditions described as: (1) incubator maintained at constant temperature of 28 +/- 1 degrees C as control; (2) temperature unregulated laboratory environment; (3) screen house; (4) grass thatched mud house and (5) corrugated iron roofed mud house. Both the grass and iron roofed mud houses were real houses found in the village communities around the ICIPE Research Centre in Mbita Point, Suba District south-western Kenya. The temperature and relative humidity of these holding environments were recorded over the study period. Mosquitoes were dissected after 24 h and 7 days to enumerate ookinetes and oocysts stages, respectively in their midguts. The mean temperature observed in the temperature-unregulated laboratory (28 degrees C) was significantly higher than the temperature of the screen house (24 degrees C) while the mean temperature observed in the iron roof mud house (27 degrees C) was comparable with that in the grass-thatched mud house (27 degrees C) although the iron roof house experienced more variation (coefficient of variation, C.V., = 9.6%) and higher peaking temperatures than the grass-thatch house. The mean relative humidity for the laboratory and screen house were 23% and 32.5%, respectively, much lower than relative humidity in the incubator (73%). Relative humidity of the grass thatch hut (42%) and Iron roof hut (51%) were also lower than those of the incubator. The ookinete intensities for mosquitoes in the screen house (10.11 +/- 1.79 ookinetes/midgut) were not statistically different (P = 0.41) from those held in the laboratory (7.50 +/- 1.19 ookinetes/midgut) or in the incubator (9.89 +/- 1.47 ookinetes/midgut). The holding environments influenced the oocyst infection rates (P = 0.04) that increased from 8.4% in the screen house to 10.2% in the laboratory. The highest infection rate (12.5%) was observed in mosquitoes held in the incubator. However, the mean oocyst intensities in mosquitoes did not differ under these environments (P = 0.58). In the 'real village house' environments, the mean ookinete intensities were not statistically different between groups of mosquitoes compared to the incubator (P = 0.86). The oocyst infection rates observed in the highly fluctuating iron roof house were 9.4% as compared to 9.0% and 6.9% in the more stable and constant habitats of grass thatch house and incubator, respectively. Results show that the natural microhabitats did not influence the infections rates in mosquitoes (P = 0.62). These findings indicate that the variation in temperatures prevailing in western Kenya particularly inside the village houses do not impede the development of malaria parasites in A. gambiae mosquitoes.

Influence of sugar availability and indoor microclimate on survival of Anopheles gambiae (Diptera: Culicidae) under semifield conditions in western Kenya.

The influence of indoor microclimate on survival of female Anopheles gambiae sensu stricto Giles (Diptera: culicidae) mosquitoes fed on different nutrition sources was evaluated in a semifield experimental hut exposed to ambient climate in western Kenya. Cages of mosquitoes (n approximately 50 per cage) were placed in nine positions within the hut combining three different sides and three different heights. At each height and side, mosquitoes were offered either human blood (once every 2 d), glucose (6% wt:vol) or a combination of the two diets over three experiments so that each cage position received one diet source. The effect of diet on survival was significant with mean survival times of 14 d for mosquitoes fed blood alone, 29 d for sugar alone and 33 d for blood plus sugar. Sugar availability decreased the odds of mortality approximately 85% compared with the blood group. Micro heterogeneities of temperature but not relative humidity also influenced survival although to a much lesser extent. The side but not height within the hut at which mosquitoes were placed, influenced survival but could not be explained by either temperature or relative humidity differences. The potential influence of seemingly minor heterogeneities of indoor microclimate upon vector longevity and vectorial capacity may merit further investigation. Also, the availability of sugar was shown to be a potentially crucial determinant of vectorial capacity. Compared with blood alone, the availability of sugar served to increase survival potential of vectors beyond ages at which they are old enough to transmit malaria.

Resistance of early midgut stages of natural Plasmodium falciparum parasites to high temperatures in experimentally infected Anopheles gambiae (Diptera: Culicidae).

We studied the effects of high temperature, 30 and 32 versus 27 C on early Plasmodium falciparum development in Anopheles gambiae experimentally infected with gametocytes from 30 volunteers with mean density of 264.1 gametocytes/microl blood (range: 16-1,536/microl). From several batches of mosquitoes, fed by membrane feeding, midguts of individual mosquitoes were dissected at 24 hr for ookinete enumeration and at 7 days to quantify oocysts. There were temperature-related differences in mean ookinete intensity per mosquito midgut, with 9.71 +/- 1.6 at 27 C, 9.85 +/- 2.32 at 30 C, and 3.89 +/- 0.81 at 32 C. The prevalence of oocyst infection decreased with an increase in temperatures from 15.9 to 8.5 to 6.4% at 27, 30, and 32 C, respectively. The average oocyst intensities for the infected mosquitoes increased with temperatures from 2.9 at 27 C to 3.5 at 30 C, and to 3.3 at 32 C. However, the success of infections was reduced at 30 and 32 C, and resulted in greater losses during consecutive inter-stage parasite development. The most significant impact of high temperatures occurred at the transition between macrogametocytes and ookinetes, whereas the transition between ookinetes and oocysts apparently was not affected. In contrast to other reports, exposure of mosquitoes infected with natural parasites to high temperatures did not eliminate preoocyst stages, as has been observed from laboratory studies using the NF-54 strain of P. falciparum. This observation of parasite resistance to high temperatures is consistent with the natural situation in tropical environments where perennial malaria transmission occurs during hot dry seasons.

Influence of age and previous diet of Anopheles gambiae on the infectivity of natural Plasmodium falciparum gametocytes from human volunteers.

The effect of age and dietary factors of Anopheles gambiae (Diptera: Culicidae) on the infectivity of natural Plasmodium falciparum parasites was studied. Mosquitoes of various ages (1-3, 4-7 and 8-11 day old) and those fed blood (either single or double meals) and sugar meals were experimentally co-infected with P. falciparum gametocytes obtained from different naturally infected human volunteers. On day 7, midguts were examined for oocyst infection to determine whether mosquito age or diets have significant effects on parasite infectivity. The age of the mosquitoes did not significantly influence the oocyst infection rates (chi2 = 48.32, df = 40, P = 0.172) or oocyst load (# of oocysts/midgut) (P = 0.14) observed. Oocyst load between groups was not significantly different. Similarly, the type of diet (either blood or sugar) did not influence oocyst infection rates (chi2 = 16.52, df = 19, P = 0.622). However, an increase in oocyst infection rates resulted after previous feeding on double blood meals (35%) compared to single blood meals (25%), with comparable oocyst load. These observations are in agreement with those reported in previous studies suggesting that increased mosquito nutritional reserves resulting from increased dietary resources is favorable for malaria infectivity. This field-based study indicates that vector competence of An. gambiae to natural P. falciparum parasites does not vary with age and that nutritional resources acquired prior to an infectious blood meal plays a crucial role in mosquito-parasite relationships.

Mosquito feeding assays to determine the infectiousness of naturally infected Plasmodium falciparum gametocyte carriers.

INTRODUCTION: In the era of malaria elimination and eradication, drug-based and vaccine-based approaches to reduce malaria transmission are receiving greater attention. Such interventions require assays that reliably measure the transmission of Plasmodium from humans to Anopheles mosquitoes. METHODS: WE COMPARED TWO COMMONLY USED MOSQUITO FEEDING ASSAY PROCEDURES: direct skin feeding assays and membrane feeding assays. Three conditions under which membrane feeding assays are performed were examined: assays with i) whole blood, ii) blood pellets resuspended with autologous plasma of the gametocyte carrier, and iii) blood pellets resuspended with heterologous control serum. RESULTS: 930 transmission experiments from Cameroon, The Gambia, Mali and Senegal were included in the analyses. Direct skin feeding assays resulted in higher mosquito infection rates compared to membrane feeding assays (odds ratio 2.39, 95% confidence interval 1.94-2.95) with evident heterogeneity between studies. Mosquito infection rates in membrane feeding assays and direct skin feeding assays were strongly correlated (p<0.0001). Replacing the plasma of the gametocyte donor with malaria naïve control serum resulted in higher mosquito infection rates compared to own plasma (OR 1.92, 95% CI 1.68-2.19) while the infectiousness of gametocytes may be reduced during the replacement procedure (OR 0.60, 95% CI 0.52-0.70). CONCLUSIONS: Despite a higher efficiency of direct skin feeding assays, membrane feeding assays appear suitable tools to compare the infectiousness between individuals and to evaluate transmission-reducing interventions. Several aspects of membrane feeding procedures currently lack standardization; this variability makes comparisons between laboratories challenging and should be addressed to facilitate future testing of transmission-reducing interventions.

Spatial and temporal distribution patterns of Anopheles arabiensis breeding sites in La Reunion Island--multi-year trend analysis of historical records from 1996-2009.

BACKGROUND: An often confounding facet of the dynamics of malaria vectors is the aquatic larval habitat availability and suitable conditions under which they can thrive. Here, we investigated the impact of environmental factors on the temporal and spatial distribution of larval habitats of Anopheles arabiensis in different locations on La Reunion Island. METHODS: A retrospective examination was made from archival data which provided the complete enumeration of An. arabiensis breeding habitats in three distinct geographic zones--extending North-east, West and South of the island over 14 years, from January 1996 to December 2009. Data on the occurrence and the number of active larval habitats at each of a total of 4376 adjacent ellipsoid grid cells (216,506 square meters each) were used (1) to provide the geographic extent of breeding site availability from year to year and (2) to analyze associations with prevailing environmental factors, habitat types, and locations. RESULTS: Anopheles arabiensis utilized a spectrum of man-made and natural aquatic habitats, most of which were concentrated primarily in the rock pools located in ravines and river fringes, and also in the large littoral marshes and within the irrigated agricultural zones. The numbers of breeding site per sampling grid differed significantly in different parts of the island. In contrast to an originally more widespread distribution across the island in the 1950s, detailed geographic analyses of the data obtained in the period extending from 1996-2009 showed an intriguing clustered distribution of active breeding sites in three discontinuous geographic zones, in which aquatic habitats availability fluctuates with the season and year. Seasonality in the prevalence of anopheles breeding sites suggests significant responsiveness to climatic factors. CONCLUSIONS: The observed retreat of An. arabiensis distribution range to lower altitudinal zones (< 400 m) and the upward shift in the most remote littoral areas in the northeast and southwest regions suggest the possible influence of biogeographic factors, changes in land use and control operations. The results of this study would allow for a more rational implementation of control strategies across the island.

Human immune responses that reduce the transmission of Plasmodium falciparum in African populations.

Malaria-infected individuals can develop antibodies which reduce the infectiousness of Plasmodium gametocytes to biting Anopheles mosquitoes. When ingested in a bloodmeal together with gametocytes, these antibodies reduce or prevent subsequent parasite maturation in the insect host. This transmission-blocking immunity is usually measured in human sera by testing its effect on the infectivity of gametocytes grown in vitro. Here we evaluate evidence of transmission-blocking immunity in eight studies conducted in three African countries. Plasmodium falciparum gametocytes isolated from each individual were fed to mosquitoes in both autologous plasma collected with the parasites, and permissive serum from non-exposed donors. Evidence of transmission reducing effects of autologous plasma was found in all countries. Experiments involving 116 Gambian children (aged 0.5-15 years) were combined to determine which factors were associated with transmission reducing immune responses. The chances of infecting at least one mosquito and the average proportion of infected mosquitoes were negatively associated with recent exposure to gametocytes and sampling late in the season. These results suggest that effective malaria transmission-reducing antibodies do not commonly circulate in African children, and that recent gametocyte carriage is required to initiate and/or boost such responses.

Submicroscopic gametocytes and the transmission of antifolate-resistant Plasmodium falciparum in Western Kenya.

BACKGROUND: Single nucleotide polymorphisms (SNPs) in the dhfr and dhps genes are associated with sulphadoxine-pyrimethamine (SP) treatment failure and gametocyte carriage. This may result in enhanced transmission of mutant malaria parasites, as previously shown for chloroquine resistant parasites. In the present study, we determine the association between parasite mutations, submicroscopic P. falciparum gametocytemia and malaria transmission to mosquitoes. METHODOLOGY/PRINCIPAL FINDINGS: Samples from children treated with SP alone or in combination with artesunate (AS) or amodiaquine were genotyped for SNPs in the dhfr and dhps genes. Gametocytemia was determined by microscopy and Pfs25 RNA-based quantitative nucleic acid sequence-based amplification (Pfs25 QT-NASBA). Transmission was determined by membrane-feeding assays. We observed no wild type infections, 66.5% (127/191) of the infections expressed mutations at all three dhfr codons prior to treatment. The presence of all three mutations was not related to higher Pfs25 QT-NASBA gametocyte prevalence or density during follow-up, compared to double mutant infections. The proportion of infected mosquitoes or oocyst burden was also not related to the number of mutations. Addition of AS to SP reduced gametocytemia and malaria transmission during follow-up. CONCLUSIONS/SIGNIFICANCE: In our study population where all infections had at least a double mutation in the dhfr gene, additional mutations were not related to increased submicroscopic gametocytemia or enhanced malaria transmission. The absence of wild-type infections is likely to have reduced our power to detect differences. Our data further support the use of ACT to reduce the transmission of drug-resistant malaria parasites.

Moderate effect of artemisinin-based combination therapy on transmission of Plasmodium falciparum.

Background. Artemisinin-based combination therapy (ACT) reduces microscopically confirmed gametocytemia and mosquito infection. However, molecular techniques have recently revealed high prevalences of submicroscopic gametocytemia. Our objective here was to determine the effect of sulfadoxine-pyrimethamine (SP) monotherapy and treatment with SP plus amodiaquine (AQ), SP plus artesunate (AS), and artemether-lumefantrine (AL; Coartem) on submicroscopic gametocytemia and infectiousness.Methods. Kenyan children (n=528) 6 months-10 years of age were randomized to 4 treatment arms. Gametocytemia was determined by both microscopy and Pfs25 RNA-based quantitative nucleic acid sequence-based amplification (Pfs25 QT-NASBA). Transmission was determined by membrane-feeding assays.Results. Gametocyte prevalence, as determined by Pfs25 QT-NASBA, was 89.4% (219/245) at enrollment and decreased after treatment with SP plus AS, SP plus AQ, and AL. Membrane-feeding assays for a group of randomly selected children revealed that the proportion of infectious children was as much as 4-fold higher than expected when based on microscopy. ACT did not significantly reduce the proportion of infectious children but did reduce the proportion of infected mosquitoes.Conclusions. Submicroscopic gametocytemia is common after treatment and contributes considerably to mosquito infection. Our findings should be interpreted in the context of transmission intensity, but the effect of ACT on malaria transmission appears to be moderate and restricted to the duration of gametocyte carriage and the proportion of mosquitoes that are infected by carriers.