Submicroscopic Plasmodium falciparum gametocyte carriage is common in an area of low and seasonal transmission in Tanzania

OBJECTIVE: Recently developed molecular gametocyte detection techniques have shown that submicroscopic Plasmodium falciparum gametocytes are common in symptomatic patients and can infect mosquitoes. The relevance for the infectious reservoir of malaria in the general population remains unknown. In this study, we investigated submicroscopic asexual parasitaemia and gametocytaemia in inhabitants of an area of hypoendemic and seasonal malaria in Tanzania. METHODS: Two cross-sectional malariometric surveys were conducted in the dry and wet seasons of 2005 in villages in lower Moshi, Tanzania. Finger prick blood samples were taken to determine the prevalence of P. falciparum parasites by microscopy, rapid diagnostic test and real-time nucleic acid sequence-based amplification (QT-NASBA). RESULTS: 2752 individuals participated in the surveys, of whom 1.9% (51/2721) had microscopically confirmed asexual parasites and 0.4% (10/2721) had gametocytes. In contrast, QT-NASBA revealed that 32.5% (147/453) of the individuals harboured asexual parasites and 15.0% (68/453) had gametocytes. No age dependency or seasonality was observed in submicroscopic parasite carriage. DISCUSSION: Molecular detection techniques reveal that carriage of submicroscopic asexual parasite and gametocyte densities is relatively common in this low transmission area. Submicroscopic gametocytaemia is likely to be responsible for maintaining malarial transmission in the study area.     

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.     

Population biology and antimalarial resistance: The transmission of antimalarial drug resistance in Plasmodium falciparum

Malaria morbidity and mortality continue to increase across sub-Saharan Africa. This is largely as a result of the continued use of chloroquine and sulfadoxine-pyrimethamine, despite widespread resistance. Although eliminating the asexual stages of Plasmodium falciparum is the focus of treatment of individual symptomatic patients, at a population level, reducing the carriage of gametocytes - the sexual stage responsible for infection of the mosquito vector - is necessary to limit the transmission of malaria parasites and the spread of antimalarial resistance. The probability of a mosquito being infected depends on the prevalence, duration and density of viable gametocyte carriage in the human host, although additional humoral and leukocyte factors also affect transmissibility. There is a log-sigmoid relationship between gametocyte density in the patients' blood and infectivity to the mosquito. The infectivity and thus transmission potential associated with a particular antimalarial treatment can be characterised as a function of blood gametocyte density and time, summing these over the acute and all subsequent recrudescences of that infection. Gametocyte carriage and infectivity to mosquitoes is consistently higher in patients infected with drug resistant compared with drug sensitive malaria parasites. It is the ratio of transmission potential in drug resistant versus sensitive infections that drives the spread of resistance. Early access to highly effective antimalarial treatment reduces the risk of disease progression and limits gametocyte carriage. The remarkable spread of sulfadoxine-pyrimethamine (SP) resistance across vast regions results from the very high post-treatment prevalence and density of gametocyte carriage following SP treatment. In areas of low intensity malaria transmission, the gametocyte-reducing effect of widespread use of artemisinin-based combination therapy has resulted in a sustained decrease in malaria transmission and a decrease in the spread of resistance. Malaria treatment policy should be based primarily on therapeutic efficacy against asexual stages, but should also consider transmission reduction potential. Artemisinin-based combination therapies are the only antimalarials currently available which rapidly reduce both asexual and gametocyte stages of the P. falciparum lifecycle.     

Dynamics of P. falciparum gametocytemia in symptomatic patients in an area of intense perennial transmission in Tanzania

We investigated the dynamics of Plasmodium falciparum gametocytemia in symptomatic patients attending a local dispensary in the Kilombero district, Tanzania. Consenting individuals aged one and above, with varying asexual and sexual parasitemias were treated appropriately and asked to return weekly for 28 days. Gametocyte prevalence was highest on Day 7 of follow-up in all age groups (overall 30.5%). Multifactorial analysis showed that young age (chi2 = 18.4; P = 0.004), high asexual parasitemia on presentation (chi2 = 19.4; P = 0.0007) and gametocyte positivity on presentation (chi2 = 29.4; P = 0.001) were all significantly associated with the presence of gametocytes on Days 7 and 14 of follow-up. High presentation of asexual parasitemia alone was positively correlated with higher gametocyte densities on both days of follow-up (F4, 297 = 2.0; P = 0.049). Gametocyte incidence rates decreased significantly with age (chi2 = 7.6, P < 0.005). In summary, in this group of chloroquine-treated individuals, gametocyte prevalence and incidence rates decreased with age, while densities remained relatively constant.     

Effects of chloroquine, amodiaquine and pyrimethamine-sulfadoxine on Plasmodium falciparum gametocytemia

The effects of chloroquine, amodiaquine and pyrimethamine-sulfadoxine (SP) (Fansidar) on the infection rate and density of Plasmodium falciparum gametocytes were studied in 198 patients with falciparum malaria from an area in the Punjab where malaria is endemic but seasonally transmitted. One month following treatment of 100 patients, SP had reduced the gametocyte carrier rate from 37% to 6% and the mean gametocyte density from 80 to 1.4 per mm3 of blood. Chloroquine and amodiaquine were much less effective. Since SP has no gametocytocidal properties and the reduction in gametocytes coincided with clearance of asexual parasitemias, gametocytes were probably reduced subsequent to the cure of the asexual malaria infections. If used during the nontransmission season, SP might be an effective component of an integrated program for reducing malaria transmission in the Punjab and other areas where 4-aminoquinoline-resistant and SP-sensitive falciparum malaria exists.     

Treatment failure of pyrimethamine-sulphadoxine and induction of Plasmodium falciparum gametocytaemia in children in western Kenya

Sub-Saharan Africa faces increasing levels of resistance of Plasmodium falciparum parasites to the first-line drug pyrimethamine-sulphadoxine (SP). Successful treatment with SP is reported to induce gametocytes and drug resistance may further increase gametocytaemia after treatment. Treatment success, gametocyte prevalence and gametocyte density were determined in 224 asymptomatic children in western Kenya on day 7 after treatment with SP. Treatment failure (R2 or R3 resistance) was observed in 22% of the children. The relative risk to show gametocytes on day 7 after treatment in children with treatment failure was 4.1 (95% CI 1.4-11.6) times higher compared to children with a sensitive infection, after adjustment for age and trophozoite density at the start of treatment. In addition, the gametocyte density was also higher upon SP treatment failure. These findings are reason for concern, as the increased gametocyte prevalence and density after SP treatment failure may increase the spread of SP-resistant strains in the population.     

Increased gametocytemia after treatment: an early parasitological indicator of emerging sulfadoxine-pyrimethamine resistance in falciparum malaria

BACKGROUND: Although malaria treatment aims primarily to eliminate the asexual blood stages that cause illness, reducing the carriage of gametocytes is critical for limiting malaria transmission and the spread of resistance. METHODS: Clinical and parasitological responses to the fixed-dose combination of sulfadoxine and pyrimethamine in patients with uncomplicated falciparum malaria were assessed biannually since implementation of this treatment policy in 1998 in Mpumalanga Province, South Africa. RESULTS: Despite sustained cure rates of > 90% (P = .14), the duration of gametocyte carriage increased from 3 to 22 weeks (per 1000 person-weeks) between 1998 and 2002 (P < .001). The dhfr and dhps mutations associated with sulfadoxine-pyrimethamine resistance were the most important drivers of the increased gametocytemia, although these mutations were not associated with increased pretreatment asexual parasite density or slower asexual parasite clearance times. The geometric mean gametocyte duration and area under the gametocyte density time curve (per 1000 person-weeks) were 7.0 weeks and 60.8 gametocytes/microL per week, respectively, among patients with wild-type parasites, compared with 45.4 weeks (P = .016) and 1212 gametocytes/microL per week (P = .014), respectively, among those with parasites containing 1-5 dhfr/dhps mutations. CONCLUSIONS: An increased duration and density of gametocyte carriage after sulfadoxine-pyrimethamine treatment was an early indicator of drug resistance. This increased gametocytemia among patients who have primary infections with drug-resistant Plasmodium falciparum fuels the spread of resistance even before treatment failure rates increase significantly.     

A new model for testing gametocytocidal effects of some antimalarial drugs on Plasmodium falciparum in vitro.

A technique is described for obtaining pure gametocyte cultures of Plasmodium falciparum, using pyrimethamine at the minimum concentration for inhibition of asexual parasites. Routine cultures producing sexual stages were exposed to pyrimethamine on days 5 and 6. These cultures grew synchronously and contained gametocytes of stages II, III and V on day 7, 9 and 15 of the cultures respectively. The pyrimethamine-treated gametocytes were more infective to mosquitoes than were untreated controls. This model for the culture of pure gametocytes was used to observe the activity of chloroquine, halofantrine, pyrimethamine and quinine on the gametocyte stage III of Plasmodium falciparum strain NF54 in vitro. NF54 was shown to be sensitive to chloroquine, quinine and pyrimethamine, but the results showed that halofantrine was the most effective drug in reducing the number of gametocytes. A concentration of 3 x 10(-9) M halofantrine was lethal to both asexual parasites and gametocytes. The gametocytocidal EC90 of chloroquine (1 x 10(-6) M) and that of quinine (9 x 10(-7) M) were equal to the minimum inhibitory concentration of asexual stages of isolates of P. falciparum considered as highly resistant to these drugs. A high concentration of pyrimethamine (1 x 10(-4) M) had, in contrast, little effect on gametocytes.     

Seasonal patterns of Plasmodium falciparum gametocyte prevalence and density in a rural population of Burkina Faso

Gametocytes are the malaria parasite stages that secure the transmission from the human host to the mosquito. The identification of natural parameters that influence gametocyte carriage can contribute to a better understanding of the dynamics of the sexual stage parasites for transmission reducing strategies. A total of 3400 blood slide readings were done during four cross-sectional surveys (2002-2003) including all age groups to determine the effect of season on Plasmodium falciparum gametocytes in a seasonal malaria transmission area of Burkina Faso. Entomological data were collected to determine the malaria transmission intensity in relation to seasons. Transmission intensity was estimated by monthly EIRs, averaging 28 and 32 infective bites/person/month in the wet seasons of 2002 and 2003, respectively. The EIR in the dry seasons was below one infective bite/person/month. The gametocyte prevalence was significantly higher at the start and peak of the wet season compared to the dry season when corrected for asexual parasite density and age. Gametocyte density significantly increased during the wet season after correction for asexual parasite density and age. In this study, season appears to be an independent parameter that determines gametocyte prevalence and density and should be considered to be included in epidemiological studies on malaria transmission.     

Long term primaquine administration to reduce Plasmodium falciparum gametocyte transmission in hypoendemic areas

Artemesinin-combination therapies (ACTs) for falciparum malaria reduce gametocyte carriage, and therefore reduce transmission. Artemisinin derivatives act only against young gametocytes, but primaquine acts against mature gametocytes (which are usually present in the circulation at the time the patient presents for treatment). Both artemisnin derivatives and primaquine have short half-lives (less than 1 hour and 8 hours, respectively). Therefore, asexual parasites and young gametocytes may remain after completing ACT. Single dose of primaquine (0.5-0.75 mg base/kg) at the end of ACT can kill only mature gametocytes (if present) but cannot kill young gametocytes (if present). Remaining asexual forms and sequestered young gametocytes remaining after completion of ACT may develop into mature gametocytes 7-15 days later. Some patients have the first appearance of gametocytemia 4-8/day after completion of ACT. Thus, additional doses of primaquine (0.5-0.75 mg base/kg) given 15-18 days after or concurrently with 3 day-ACT respectively or given 15-22 days after or concurrently with 7 day-ACT respectively may be beneficial in killing the remaining mature gametocytes and thus contribute to interruption of P. falciparum gametocyte transmission more affectively than giving only a single dose of primaquine just after completing ACT.     

Host haematological factors influencing the transmission of Plasmodium falciparum gametocytes to Anopheles gambiae s.s. mosquitoes

We investigated the relationship between selected host haematological and parasitological parameters and the density and infectivity of Plasmodium falciparum gametocytes. 143 individuals (age range 1-62 years) attending an outpatient clinic in Farafenni, The Gambia, who had peripheral blood gametocytaemia were recruited (mean gametocyte density 123.7/microl, range 5-17,000/microl). Of the parameters measured, packed cell volume (PCV), reticulocyte count (RetC) and lymphocyte count (LyC) were significantly associated with gametocyte density (r = - 0.17, P < 0.05; r = 0.21, P < 0.01; r = 0.18, P < 0.05, respectively). Data from membrane feeding experiments in which 15 or more mosquitoes were dissected showed that 60.7% (53/87) of gametocyte carriers infected one or more mosquitoes. Gametocyte density was strongly correlated with transmission success (TS) (r = 0.3, P < 0.005) and, in successful infections, with both mosquito prevalence (MP) (r = 0.36, P < 0.005) and mean oocyst burden (MOB) (r = 0.65, P < 0.0001). None of the other factors measured were significantly associated with any of these indices in bivariate analysis. Regression modelling showed that both gametocyte density and PCV were positively associated with gametocyte carrier infectivity to mosquitoes (LRchi2 = 100.7 and 47.2, respectively) and, in successful infections, with MOB (beta = 0.16, t = 4.9, P < 0.001; beta = 0.02, t = 2.3, P < 0.05, respectively). The positive association with PCV suggests that blood meal quality influences infection probably as a nutritional requirement, however, as this effect was most apparent at high gametocyte densities, its epidemiological significance is questionable. Though the haematological parameters associated with gametocyte density are a direct consequence of asexual infection, they may also represent an adaptive mechanism for optimization of sexual stage development.     

Epidemiology of Plasmodium falciparum in a rice field and a savanna area in Burkina Faso: seasonal fluctuations of gametocytaemia and malarial infectivity.

For a better understanding of the epidemiology of Plasmodium falciparum in an African savanna area, the authors have: (a) defined the real gametocyte reservoir in the native population; (b) followed the fluctuations of gametocytaemia throughout the transmission period; and (c) measured the infectiousness of malarious individuals to mosquitoes. The transversal surveys, in different villages of this endemic area, have shown that gametocyte carrier rates decreased with age and malaria experience; 10.9% of the whole population were potentially infectious to mosquitoes, and of these 73% were children and only 27% were adults. The longitudinal studies have shown that the P. falciparum gametocyte rate depends on the equilibrium between the gametocyte conversion rates and the density of the asexual forms. When there are large numbers of children who become carriers of the sexual stage of the parasite and at the same time a small number who lose their gametocyte infection, the gametocyte rate increases in the population; and vice versa. The circumstances under which gametocytes are produced are not well-known. Two factors seem to be important: the level of the parasite density and immune mechanisms. The infectiousness of malarious individuals was estimated by the 'mosquito infection probability'. The percentage of mosquitoes infected after feeding on gametocyte carriers (which may partly reflect the infectiousness of a human population to mosquitoes) was multiplied by the percentage of gametocyte carriers in the population. This indicated that, in this endemic area, 4% of biting mosquitoes would become infected; but this theoretical mosquito infection probability is over-estimated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Risk factors for gametocyte carriage in Gambian children.

A widespread reduction in Plasmodium falciparum gametocyte prevalence could reduce malaria transmission. After infection with P. falciparum, a variable proportion of people are found to be gametocytemic. We analyzed risk factors associated with gametocytemia at presentation and 7 days later. We enrolled 1,198 children in 2 antimalarial drug trials between September and December 1998. The children were assigned to 1 of 4 treatment groups: chloroquine only; pyrimethamine-sulfadoxine (PSD) only; PSD combined with 1 dose of artesunate; and PSD combined with 3 doses of artesunate. By the time of enrollment, 200 (17%) of 1,198 children were gametocyte carriers. Three independent risk factors were associated with gametocytemia at enrollment. Children with anemia were more likely to carry gametocytes, whereas children with fever (> 37.4 degrees C) or high parasite densities (> 100,000 parasites/microL) were less frequently gametocyte carriers. Children with at least 2 of the risk factors were 4 times more likely to be gametocytemic than children with < 2 risk factors (odds ratio [OR], 4.4; 95% confidence interval [CI], 2.7-7.1). Seven days after the start of treatment, 355 (37%) of 466 assessable children were found to be gametocyte carriers. Children treated with PSD alone had a significantly higher risk of being gametocytemic by Day 7 compared with children in the other 3 treatment groups. In the subgroup of children who had no detectable gametocytes on enrollment, the effect of treatment with PSD + 3 doses of artesunate was most marked. Nineteen (10%) of 198 children treated with PSD + 3 doses of artesunate became gametocytemic, in contrast to 184 (57%) of 321 children treated with PSD alone (OR, 12.7; 95% CI, 7.3-22.1). Early treatment with highly effective antimalarial therapy has the greatest chance of preventing gametocytemia. The choice of a first-line antimalarial drug for uncomplicated malaria should not only take into consideration the ablation asexual parasitemia but also the suppression of gametocytemia.     

Transmission-reducing immunity is inversely related to age in Plasmodium falciparum gametocyte carriers

Immunity to the sexual stages of Plasmodium falciparum is induced during natural infections and can significantly reduce the transmission of parasites to mosquitoes (transmission reducing activity; TRA) but little is known about how these responses develop with increasing age/exposure to malaria. Routinely TRA is measured in the standard membrane feeding assay (SMFA). Sera were collected from a total of 199 gametocyte carriers (median age 4 years, quartiles 2 and 9 years) near Ifakara, Tanzania; 128 samples were tested in the SMFA and generated TRA data classified as a reduction of > 50% and > 90% of transmission. TRA of > 50% was highest in young children (aged 1-2) with a significant decline with age (chi(2) trend = 5.79, P = 0.016) and in logistic regression was associated with prevalence of antibodies to both Pfs230 and Pfs48/45 (OR 4.03, P = 0.011 and OR 2.43 P = 0.059, respectively). A TRA of > 90% reduction in transmission was not age related but was associated with antibodies to Pfs48/45 (OR 2.36, P = 0.055). Our data confirm that antibodies are an important component of naturally induced TRA. However, whilst a similar but small proportion of individuals at all ages have TRA > 90%, the gradual deterioration of TRA > 50% with age suggests decreased antibody concentration or affinity. This may be due to decreased exposure to gametocytes, probably as a result of increased asexual and/or gametocyte specific immunity.     

Polyclonal T-cell responses to Plasmodium falciparum gametocytes in malaria nonexposed donors

Human peripheral blood T-cells mount vigorous proliferative responses to asexual stage parasites of P. falciparum regardless of whether the donor has been exposed to the parasite. Here using highly purified P. falciparum gametocytes we show that the same is also true for this stage of the parasite. Gametocytes, like immature trophozoites, preferentially activate CD4⁺ T cells. γδ T-cell activation, commonly observed in response to mature schizonts or supernatants from asexual-stage cultures, does not occur. Furthermore, the CD4⁺ T-cell blasts stimulated by gametocytes show a similar pattern of T-cell receptor variable region (TCRVβ) usage to those stimulated by asexual parasites and there is no preferential usage of TCRVβ elements. The CD4⁺ T-cell precursor frequencies for gametocyte and asexual trophozoite responses are remarkably similar. 'Cross-reactivity' of gametocyte and asexual stage responses was confirmed by selective depletion of cells responding to particular stages of the parasite using the cytostatic drug cytosine arabinoside (Ara-C). These results suggest that the CD4⁺ T-cell responses from malaria nonexposed donors are common to gametocytes and asexual trophozoites.     

Infectiousness of the Human Population to Anopheles arabiensis by Direct Skin Feeding in an Area Hypoendemic for Malaria in Senegal

Direct skin feeding experiments are sensitive assays to determine human infectiousness to mosquitoes but are rarely used in malaria epidemiological surveys. We determined the infectiousness of inhabitants of a malaria hypoendemic area in Senegal. Gametocyte prevalence by microscopy was 13.5% (26 of 192). Of all individuals who were gametocyte positive, 44.4% (11 of 25) infected ≥ 1 Anopheles arabiensis mosquito and 10.8% (54 of 500) of mosquitoes became infected. Of all individuals who were gametocyte negative by microscopy, 4.3% (7 of 162) infected ≥ 1 mosquito and 0.4% (12 of 3240) of mosquitoes became infected. The 18.2% (12 of 66) of all mosquito infections was a result of submicroscopic gametocyte carriage and two individuals without asexual parasites or gametocytes by microscopy were infectious to mosquitoes. When infectivity and local demography was taken into account, children 5–14 years of age contributed 50.8% of the human infectious reservoir for malaria. Adults and submicroscopic gametocyte carriers may contribute considerably to onward malaria transmission in our setting.     

Gametocytemia and infectivity to mosquitoes of patients with uncomplicated Plasmodium falciparum malaria attacks treated with chloroquine or sulfadoxine plus pyrimethamine

Plasmodium falciparum gametocytemia and its related infectivity for mosquitoes was studied in 115 patients (median age = 18 years, range = 4-45) with simple malaria attacks who lived in the hypoendemic area of Dakar, Senegal. Patients were included in a 28-day in vivo sensitivity test after treatment with chloroquine (CQ, n = 82) or sulfadoxine plus pyrimethamine (SP, n = 33). The prevalence of resistant infections was 58.5% in those treated with CQ and 0% in those treated with SP. The gametocytemia peaked at day 7 after treatment. The maximal gametocyte prevalence was 38.2% in the CQ-sensitive infection group, 89.6% in the CQ-resistant group, and 97.0% in those treated with SP The maximal geometric mean gametocytemia was 2.19/microl in the CQ-sensitive infection group, 29.12/microl in the CQ-resistant group and 85.55/microl in those treated with SP. The period between appearance of the first clinical symptom and treatment was positively related to gametocyte prevalence at days 0 and 2. Experimental infection of wild Anopheles arabiensis using membrane feeders was performed at days 0 and 7, and mosquito infectivity was measured by oocyst detection on the midgut. At day 0, 14.1% of the patients had infected at least 1 mosquito, and at day 7, this value was 38.5%. The mean percentage of infected mosquitoes was 3.2% at day 0 and 12.6% at day 7. At day 7 after treatment with CQ, the relative risk for patients with resistant infections of infecting anophelines was 4.07 higher than in those with sensitive infections. No difference was observed in infectivity for mosquitoes between RI-type resistance and the RII + RIII-type resistance. A sporonticidal effect of SP was observed at day 7 after treatment. These data show that P. falciparum gametocytes and their infectivity for mosquitoes were differentiated according to the drug used, its efficacy, and the duration of symptoms before treatment; they were not dependent on the density of asexual stages. Prompt treatment of malaria cases performed at the beginning of symptoms could limit the spread of resistant parasites.     

Short report: Quantification of Plasmodium falciparum gametocytes by magnetic fractionation

A method of gametocyte quantitation in human blood was developed based on magnetic fractionation using commercially available magnetic fractionation columns (MFCs) and exploiting the magnetic susceptibility of mature Plasmodium falciparum gametocytes. The technique uses magnetic microspheres as a calibration standard. Microspheres are added to each blood sample to a known concentration. When exposed to a magnetic field, gametocytes and magnetic microspheres are preferentially captured inside MFCs. After removal of the magnetizing field, the magnetically captured material can be eluted, placed on a microscope slide that is stained, and counted by using conventional methods. The limits of quantitation for P. falciparum gametocytes were determined from serial dilutions of blood samples with known gametocyte density. The upper limit was 1,000 gametocytes/μL. Quantitative analysis above this threshold is difficult because of an over-abundance of gametocytes. The lower limit was 0.1 gametocytes/μL, and there is a significant probability of a false-negative result below this level.     

Observations on the periodicity of Plasmodium falciparum gametocytes in natural human infections

The circadian periodicity of Plasmodium falciparum gametocytes in peripheral blood was analysed in a group of children from an holoendemic community of north-eastern Tanzania. No periodicity was observed with asexual stage parasites. Gametocytes were shown to display a diurnal subperiodic pattern with a periodicity index of 31. 8. Mathematical analysis of the data indicated that P. falciparum gametocytes tend to display periodicity with a peak (15:30-19:30 h) that do not coincide with the peak (00:30-03:30 h) biting activity of the local vector, Anopheles gambiae. We were thus able to show a P. falciparum gametocyte periodicity with a harmonic wave pattern, but its functional biological significance if any, is currently unknown.