Inhibitory effects of 9-anilinoacridines on Plasmodium falciparum gametocytes

Two gametocyte-producing isolates of Plasmodium falciparum, KT1 arid KT3, were cultivated in vitro. On day 11 of cultivation, pure gametocytes containing stage II, III and IV were used to test the gametocytocidal activity of 9-anilinoacridines that had previously demonstrated their activity against the asexual stage of the parasite. After drug exposure for 48 h, gametocytes were maintained without drugs for another 2 days before thin films were prepared for parasite counting. Gametocytocidal activities of 13 analogs of 9-anilinoacridine were observed with 50% inhibitory concentrations in the range of 0.6 microM to> 100 microM The most active compound was 1'-CH2NMe2-9-anilinoacridine. Anilinoacridine derivatives with 3,6-diamino substitution had reduced gametocytocidal activity in contrast to their enhancing effect against the asexual forms. Morphological abnormalities of gametocytes were observed following drug exposure.     

Short report: effects of pyronaridine on gametocytes in patients with acute uncomplicated falciparum malaria.

The effects of pyronaridine and chloroquine on mature Plasmodium falciparum gametocytes were compared in 161 patients treated with chloroquine or pyronaridine. Neither pyronaridine nor chloroquine showed gametocytocidal activity. The relative risks of post-treatment gametocytemia after pyronaridine and chloroquine treatment in the presence of chloroquine-resistant isolates were 1.25 and 11.5, respectively, suggesting that the use of chloroquine was associated with a high risk of favoring post-therapeutic gametocytemia in chloroquine-resistant infections.     

Emergence and clearance of gametocytes in uncomplicated Plasmodium falciparum malaria

We reviewed the records of 1,175 patients with uncomplicated Plasmodium falciparum malaria to determine the prevalence of gametocytemia. All patients were admitted and received artemisinin combination therapy. Blood films were checked daily until discharge. Circulating gametocytes were observed in 240 (20.2%) of patients and in most cases (222 of 240, 92.5%) gametocytemia was detected during the first 24 hours after admission. Gametocytes were first seen in 174 cases on admission, in 24 cases at 12 hours, and in 24 cases at 24 hours. The longest interval between admission and first appearance of gametocytes was 192 hours. The median gametocyte clearance time was 163 hours (range = 12-806) in the 219 patients in whom gametocytemia resolved. However, 21 patients (9.8%) still had gametocytemia on discharge. Gametocytemia generally is present within the first 24 hours after admission, and emerges in only 1.9% of patients later on during treatment with artemisinin.     

Short report: Dynamics of Plasmodium falciparum malaria after sub-optimal therapy in Uganda

We followed parasite genotypes of 75 patients for 42 days after treatment of uncomplicated malaria with chloroquine + sulfadoxine-pyrimethamine in Kampala, Uganda. Infections were complex (mean, 2.88 strains) and followed three patterns: 27% of patients eliminated all strains and remained parasite-free, 48% had a long aparasitemic interval followed by reappearance of original strains after 3-33 days (mean, 9.2 days), and 25% failed to clear original strains and required therapy after 3-35 days (mean, 17 days). These results highlight the complexity of malaria in Africa and have implications for efficacy trials, because missing late reappearances of strains could lead to misclassification of outcomes.     

Evaluation of sporontocidal compounds using Plasmodium falciparum gametocytes produced in vitro

A test system that uses infective gametocytes from in vitro cultures was developed for evaluating the sporontocidal activity of antimalarial compounds. In evaluating the system, pyrimethamine and cycloguanil (dihydrofolate reductase inhibitors) and primaquine (8-aminoquinoline) were tested against pyrimethamine-sensitive and pyrimethamine-resistant strains of Plasmodium falciparum. The drugs were administered to Anopheles either in a blood meal containing infective gametocytes or in a noninfective meal 2-4 days later. The mosquitoes were dissected 9-10 days after they received the infective blood meal, and the sporontocidal effect of the drugs was evaluated by the number of oocysts found in the gut. Both cycloguanil and pyrimethamine had marked sporontocidal activity. The susceptibility pattern of the strains to the sporontocidal effect of pyrimethamine and cycloguanil was similar to the susceptibility of their asexual blood stages in vitro to the schizontocidal effect of the compounds. The sporontocidal effect was observed only when the compounds were administered at the same time as the infective blood meal, but not when they were given 2-4 days later. No sporontocidal activity was observed with primaquine. This system permits more reliable quantitative observations than have been possible with previous methods.     

Parasite infectivity and immunity to Plasmodium falciparum gametocytes in Gambian children

Immunity to the sexual stages of Plasmodium falciparum can be induced during natural infections. Characterization of this immunity may facilitate the design of a transmission-blocking vaccine (TBV). This study aimed to assess the prevalence and serological correlates of functional transmission-blocking immunity in Gambian children (aged 1-4 years old) who were P. falciparum gametocyte carriers. Serological assays showed 100% response to fixed, whole parasites but only 42% to live gametes. Responses to the antigens Pfs230 and Pfs48/45 were 54.1% and 37.3%, respectively, in an IgG1 ELISA. 14/55 sera were capable of reducing the infectivity of laboratory isolate NF54 in a standard membrane-feeding assay (SMFA). This activity was strongly correlated with IgG1 responses to Pfs48/45 (r = 0.49, P < 0.001) and to a serological reaction with epitopes of the same molecule (r = 0.38, P = 0.003). A weaker correlation was observed with IgG1 to Pfs230 (r = 0.29, P = 0.03). In direct membrane feeding assays (DMFA) with autologous isolates, sera from 4/29 children showed transmission-blocking activity. There was no correlation with serological assays and the DMFA or between the SMFA and DMFA. This may be caused by variation in sexual stage antigens and/or alternative modes of transmission-blocking immunity, both of which have implications for vaccine implementation.     

Short report: Phylogenetic relationships of the anthropophilic Plasmodium falciparum malaria vectors in Africa

Malaria kills more than one million people a year, and understanding the historical association between its most notorious causative agent, Plasmodium falciparum, and its mosquito vectors is important in fighting the disease. We present a phylogenetic analysis of a number of species within the mosquito subgenus Cellia based on a selection of mitochondrial and nuclear genes. Although some of these relationships have been estimated in other studies, generally few species were included and/or statistical support at many nodes was low. Here we include two additional species of anthropophilic P. falciparum malaria vectors and reanalyze these relationships using a Bayesian method that allows us to simultaneously incorporate different models of evolution. We report data that indicate a paraphyletic relationship between five anthropophilic African mosquito vectors. Such a relationship suggests that these species can serve as independent natural experiments for anopheline immunologic responses to regular, prolonged contact with P. falciparum.     

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.     

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.     

Short report: hepatitis b infection and severe Plasmodium falciparum malaria in Vietnamese adults

We investigated the prevalence of infection with hepatitis B virus among adult Vietnamese patients hospitalized for severe Plasmodiumfalciparum malaria. Sera from patients admitted with severe malaria in Ho Chi Minh City, Vietnam, between May 1991 and January 1996 were assayed for hepatitis B surface antigen (HB(s)Ag) by a commercial enzyme-linked immunosorbent assay kit. The overall prevalence of HB(s)Ag was 23.77% (77 of 324). This was higher than reported estimates of prevalence in the general catchment population for the study hospital (mean, 9.8%; range, 9-16%). No association was found between risk of death caused by severe malaria and HB(s)Ag. Patients admitted with cerebral malaria had a slightly greater risk of registering positive for HB(s)Ag (relative risk, 1.28; 95% confidence interval, 1.04-1.58) relative to other manifestations of severe malaria. Chronic infection with hepatitis B virus may be a risk factor for severe malaria.     

Short report: Positive correlation between rosetting and parasitemia in Plasmodium falciparum clinical isolates

Plasmodium falciparum isolates that form rosettes with uninfected red cells are associated with severe malaria in African children, although the mechanism by which rosetting contributes to severe disease is unknown. Here we have analyzed the relationship between rosetting and parasitemia in two samples of clinical isolates from children with malaria in Kilifi, Kenya. A consistent positive correlation was found between rosetting and parasitemia (Spearman's rank correlation coefficent p = 0.467, P < .001, n = 154, for 1993 study; p = .407, P < .001, n = 74, for 2000 study). Rosetting may enhance parasite growth and survival by facilitating invasion or promoting immune evasion, thus allowing higher parasitemia to develop and increasing the likelihood of severe malaria.     

Short report: entomologic inoculation rates and Plasmodium falciparum malaria prevalence in Africa.

Epidemiologic patterns of malaria infection are governed by environmental parameters that regulate vector populations of Anopheles mosquitoes. The intensity of malaria parasite transmission is normally expressed as the entomologic inoculation rate (EIR), the product of the vector biting rate times the proportion of mosquitoes infected with sporozoite-stage malaria parasites. Malaria transmission intensity in Africa is highly variable with annual EIRs ranging from < 1 to > 1,000 infective bites per person per year. Malaria control programs often seek to reduce morbidity and mortality due to malaria by reducing or eliminating malaria parasite transmission by mosquitoes. This report evaluates data from 31 sites throughout Africa to establish fundamental relationships between annual EIRs and the prevalence of Plasmodium falciparum malaria infection. The majority of sites fitted a linear relationship (r2 = 0.71) between malaria prevalence and the logarithm of the annual EIR. Some sites with EIRs < 5 infective bites per year had levels of P. falciparum prevalence exceeding 40%. When transmission exceeded 15 infective bites per year, there were no sites with prevalence rates < 50%. Annual EIRs of 200 or greater were consistently associated with prevalence rates > 80%. The basic relationship between EIR and P. falciparum prevalence, which likely holds in east and west Africa, and across different ecologic zones, shows convincingly that substantial reductions in malaria prevalence are likely to be achieved only when EIRs are reduced to levels less than 1 infective bite per person per year. The analysis also highlights that the EIR is a more direct measure of transmission intensity than traditional measures of malaria prevalence or hospital-based measures of infection or disease incidence. As such, malaria field programs need to consider both entomologic and clinical assessments of the efficacy of transmission control measures.     

Short report: in vivo sensitivity of Plasmodium falciparum to halofantrine in southern central Vietnam

Drug-resistant Plasmodium falciparum is present in Vietnam. We assessed the in vivo sensitivity of P. falciparum to halofantrine in two villages in the southern part of central Vietnam. Halofantrine (8 mg/kg x 3 doses) was administered to 37 patients with either P. falciparum (n = 32) or mixed P. falciparum/P. vivax malaria (n = 5). End points were parasite sensitivity or resistance (RI/RII/RIII) determined by parasite clearance, persistence, or recurrence during 28 days of follow-up. By day 28, 31 (93.9%) of 33 (95% confidence interval = 79.8-99.2%) patients were sensitive. Two patients had recurrent P. falciparum parasitemia on days 14 and 21. Halofantrine effectively treated uncomplicated P. falciparum malaria in these Vietnamese patients.     

Short report: lack of prediction of amodiaquine efficacy in treating Plasmodium falciparum malaria by in vitro tests

Amodiaquine (AQ) is currently a major candidate for new antimalarial combinations, although in vivo and in vitro tests have been rarely simultaneously investigated. The efficacy of AQ was assessed at the dose of 30 mg/kg in treating Plasmodium falciparum malaria attacks in 74 children from southeast Gabon, and the in vitro activity of monodesethylamodiaquine (MdAQ), the main metabolite of AQ, was measured against P. falciparum parasites isolated from these children. Treatment failures were observed in 40.5% of the children, while 5.4% of the isolates showed in vitro resistance to MdAQ. No relationship was observed between in vivo and in vitro susceptibility. The in vitro activities of MdAQ and chloroquine were correlated. The reasons for such disparities between in vivo and in vitro AQ activities are discussed and the issue of the validity of in vitro tests to measure AQ efficacy is raised.     

Patterns of antigen expression in asexual blood stages and gametocytes of Plasmodium falciparum

The stage specificity and localization of 12 Plasmodium falciparum antigens were determined by immunofluorescence using acetone-fixed parasites reacted with monospecific antibodies against cloned antigens. Antibodies were prepared by immunization of rabbits with recombinant proteins or by affinity purification of human plasma against cloned antigen adsorbents. Most of the antigens occurred predominantly in mature asexual parasites, two were abundant in ring stages and three were absent in rings. Four of the 12 antigens were detected in asexual stages but not in gametocytes. Grouping of antigens by localization within blood stages was difficult because of the complexity of fluorescence patterns observed. With some antibodies, fluorescence was apparently distributed evenly over the parasites, but in other cases label was concentrated within discrete compartments or organelles. Extraparasitic intraerythrocytic fluorescence was also observed.     

Short report: High prevalence of multidrug-resistant Plasmodium falciparum malaria in the French territory of Mayotte

A drug-resistance survey was conducted in the French territory of Mayotte in the Comorian Islands in the Indian Ocean where malaria is endemic. A high prevalence of resistant Plasmodium falciparum parasites was observed, not only to chloroquine (88%) and pyrimethamine (99%), but more surprisingly to quinine (17%), mefloquine (9%), and amodiaquine (24%). This leaves few treatment alternatives other than artemisine-mefloquine combinations. However, despite notification to French Health authorities three years ago, inadequate treatment (chloroquine plus sulfadoxine-pyrimethamine) is still used in this locality. Thus, people still die of malaria in this remote territory of France.