The development of sexual stage malaria gametocytes in a Wave Bioreactor.

BACKGROUND: Blocking malaria gametocyte development in RBCs or their fertilization in the mosquito gut can prevent infection of the mosquito vector and passage of disease to the human host. A 'transmission blocking' strategy is a component of future malaria control. However, the lack of robust culture systems for producing large amounts of Plasmodium falciparum gametocytes has limited our understanding of sexual-stage malaria biology and made vaccine or chemotherapeutic discoveries more difficult. METHODS: The Wave BioreactorTM 20/50 EHT culture system was used to develop a convenient and low-maintenance protocol for inducing commitment of P. falciparum parasites to gametocytogenesis. Culture conditions were optimised to obtain mature stage V gametocytes within 2 weeks in a large-scale culture of up to a 1 l. RESULTS: We report a simple method for the induction of gametocytogenesis with N-acetylglucosamine (10 mM) within a Wave Bioreactor. By maintaining the culture for 14-16 days as many as 100 million gametocytes (stage V) were produced in a 1 l culture. Gametocytes isolated using magnetic activated cell sorting (MACS) columns were frozen in aliquots for storage. These were revitalised by thawing and shown to retain their ability to exflagellate and infect mosquitoes (Anopheles stephansi). CONCLUSIONS: The production of gametocytes in the Wave Bioreactor under GMP-compliant conditions will not only facilitate cellular, developmental and molecular studies of gametocytes, but also the high-throughput screening for new anti-malarial drugs and, possibly, the development of whole-cell gametocyte or sporozoite-based vaccines.

Lysine acetylation in sexual stage malaria parasites is a target for antimalarial small molecules.

Therapies to prevent transmission of malaria parasites to the mosquito vector are a vital part of the global malaria elimination agenda. Primaquine is currently the only drug with such activity; however, its use is limited by side effects. The development of transmission-blocking strategies requires an understanding of sexual stage malaria parasite (gametocyte) biology and the identification of new drug leads. Lysine acetylation is an important posttranslational modification involved in regulating eukaryotic gene expression and other essential processes. Interfering with this process with histone deacetylase (HDAC) inhibitors is a validated strategy for cancer and other diseases, including asexual stage malaria parasites. Here we confirm the expression of at least one HDAC protein in Plasmodium falciparum gametocytes and show that histone and nonhistone protein acetylation occurs in this life cycle stage. The activity of the canonical HDAC inhibitors trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA; Vorinostat) and a panel of novel HDAC inhibitors on early/late-stage gametocytes and on gamete formation was examined. Several compounds displayed early/late-stage gametocytocidal activity, with TSA being the most potent (50% inhibitory concentration, 70 to 90 nM). In contrast, no inhibitory activity was observed in P. falciparum gametocyte exflagellation experiments. Gametocytocidal HDAC inhibitors caused hyperacetylation of gametocyte histones, consistent with a mode of action targeting HDAC activity. Our data identify HDAC inhibitors as being among a limited number of compounds that target both asexual and sexual stage malaria parasites, making them a potential new starting point for gametocytocidal drug leads and valuable tools for dissecting gametocyte biology.

Plasmodium falciparum gametocytes: with a view to a kill.

Drugs that kill or inhibit the sexual stages of Plasmodium in order to prevent transmission are important components of malaria control programmes. Reducing gametocyte carriage is central to the control of Plasmodium falciparum transmission as infection can result in extended periods of gametocytaemia. Unfortunately the number of drugs with activity against gametocytes is limited. Primaquine is currently the only licensed drug with activity against the sexual stages of malaria parasites and its use is hampered by safety concerns. This shortcoming is likely the result of the technical challenges associated with gametocyte studies together with the focus of previous drug discovery campaigns on asexual parasite stages. However recent emphasis on malaria eradication has resulted in an upsurge of interest in identifying compounds with activity against gametocytes. This review examines the gametocytocidal properties of currently available drugs as well as those in the development pipeline and examines the prospects for discovery of new anti-gametocyte compounds.

The frequency of malaria is similar among women receiving either lopinavir/ritonavir or nevirapine-based antiretroviral treatment.

HIV protease inhibitors (PIs) show antimalarial activity in vitro and in animals. Whether this translates into a clinical benefit in HIV-infected patients residing in malaria-endemic regions is unknown. We studied the incidence of malaria, as defined by blood smear positivity or a positive Plasmodium falciparum histidine-rich protein 2 antigen test, among 444 HIV-infected women initiating antiretroviral treatment (ART) in the OCTANE trial (A5208; ClinicalTrials.gov: NCT00089505). Participants were randomized to treatment with PI-containing vs. PI-sparing ART, and were followed prospectively for ≥48 weeks; 73% also received cotrimoxazole prophylaxis. PI-containing treatment was not associated with protection against malaria in this study population.

Modelling the dynamics of Plasmodium falciparum histidine-rich protein 2 in human malaria to better understand malaria rapid diagnostic test performance.

BACKGROUND: Effective diagnosis of malaria is a major component of case management. Rapid diagnostic tests (RDTs) based on Plasmodium falciparumhistidine-rich protein 2 (PfHRP2) are popular for diagnosis of this most virulent malaria infection. However, concerns have been raised about the longevity of the PfHRP2 antigenaemia following curative treatment in endemic regions. METHODS: A model of PfHRP2 production and decay was developed to mimic the kinetics of PfHRP2 antigenaemia during infections. Data from two human infection studies was used to fit the model, and to investigate PfHRP2 kinetics. Four malaria RDTs were assessed in the laboratory to determine the minimum detectable concentration of PfHRP2. RESULTS: Fitting of the PfHRP2 dynamics model indicated that in malaria naïve hosts, P. falciparum parasites of the 3D7 strain produce 1.4 × 10⁻¹³ g of PfHRP2 per parasite per replication cycle. The four RDTs had minimum detection thresholds between 6.9 and 27.8 ng/mL. Combining these detection thresholds with the kinetics of PfHRP2, it is predicted that as few as 8 parasites/µL may be required to maintain a positive RDT in a chronic infection. CONCLUSIONS: The results of the model indicate that good quality PfHRP2-based RDTs should be able to detect parasites on the first day of symptoms, and that the persistence of the antigen will cause the tests to remain positive for at least seven days after treatment. The duration of a positive test result following curative treatment is dependent on the duration and density of parasitaemia prior to treatment and the presence and affinity of anti-PfHRP2 antibodies.

Saquinavir inhibits the malaria parasite's chloroquine resistance transporter.

The antiretroviral protease inhibitors (APIs) ritonavir, saquinavir, and lopinavir, used to treat HIV infection, inhibit the growth of Plasmodium falciparum at clinically relevant concentrations. Moreover, it has been reported that these APIs potentiate the activity of chloroquine (CQ) against this parasite in vitro. The mechanism underlying this effect is not understood, but the degree of chemosensitization varies between the different APIs and, with the exception of ritonavir, appears to be dependent on the parasite exhibiting a CQ-resistant phenotype. Here we report a study of the role of the P. falciparum chloroquine resistance transporter (PfCRT) in the interaction between CQ and APIs, using transgenic parasites expressing different PfCRT alleles and using the Xenopus laevis oocyte system for the heterologous expression of PfCRT. Our data demonstrate that saquinavir behaves as a CQ resistance reverser and that this explains, at least in part, its ability to enhance the effects of CQ in CQ-resistant P. falciparum parasites.

An improved method for undertaking limiting dilution assays for in vitro cloning of Plasmodium falciparum parasites.

BACKGROUND: Obtaining single parasite clones is required for many techniques in malaria research. Cloning by limiting dilution using microscopy-based assessment for parasite growth is an arduous and labor-intensive process. An alternative method for the detection of parasite growth in limiting dilution assays is using a commercial ELISA histidine-rich protein II (HRP2) detection kit. METHODS: Detection of parasite growth was undertaken using HRP2 ELISA and compared to thick film microscopy. An HRP2 protein standard was used to determine the detection threshold of the HRP2 ELISA assay, and a HRP2 release model was used to extrapolate the amount of parasite growth required for a positive result. RESULTS: The HRP2 ELISA was more sensitive than microscopy for detecting parasite growth. The minimum level of HRP2 protein detection of the ELISA was 0.11 ng/ml. Modeling of HRP2 release determined that 2,116 parasites are required to complete a full erythrocytic cycle to produce sufficient HRP2 to be detected by the ELISA. Under standard culture conditions this number of parasites is likely to be reached between 8 to 14 days of culture. CONCLUSIONS: This method provides an accurate and simple way for the detection of parasite growth in limiting dilution assays, reducing time and resources required in traditional methods. Furthermore the method uses spent culture media instead of the parasite-infected red blood cells, enabling culture to continue.

Antimalarial asexual stage-specific and gametocytocidal activities of HIV protease inhibitors.

The stage-specific antimalarial activities of a panel of antiretroviral protease inhibitors (PIs), including two nonpeptidic PIs (tipranavir and darunavir), were tested in vitro against Plasmodium falciparum. While darunavir demonstrated limited antimalarial activity (effective concentration [EC(50)], >50 microM), tipranavir was active at clinically relevant concentrations (EC(50), 12 to 21 microM). Saquinavir, lopinavir, and tipranavir preferentially inhibited the growth of mature asexual-stage parasites (24 h postinvasion). While all of the PIs tested inhibited gametocytogenesis, tipranavir was the only one to exhibit gametocytocidal activity.