Behavioural responses of Phlebotomus duboscqi to plant-derived volatile organic compounds.

Phlebotomine sand flies are vectors of Leishmania parasites that cause leishmaniases. Both sexes of sand flies feed on plants primarily for sugars, although the chemical cues that mediate attraction to host plants remain largely unknown. Previously, using coupled gas chromatography-mass spectrometry, the authors identified several volatile organic compounds (VOCs) common to preferred host plants for selected Afrotropical sand flies from the Fabaceae family. Of the identified volatiles, the significance of the monoterpenes linalool oxide, ocimene and p-cymene and the benzenoid m-cresol, p-cresol in sand fly behaviour is unknown. In olfactometer assays, the authors tested these compounds singly and in blends for their attractiveness to Phlebotomus duboscqi, cutaneous leishmaniasis vector in Kenya. In dose-response assays, single compounds increased the responses of males and females over controls, but their optimum attractive doses varied between the sexes. Two five-component blends, referred to as Blend-f and Blend-m for females and males respectively, were formulated and tested in dose-response assays against 1-octen-3-ol (positive control). The results of the present study showed that males and females were significantly attracted to varying levels of the two blends. In pairwise assays, the authors evaluated the most attractive of these blends to each sex (i.e., Blend Am for male against Blend Bf for female), revealing that males were attracted to both blends at varying levels, whereas females were indifferent. The study's results demonstrate that plant-derived VOCs can be exploited for sand fly management.

Effects of Irrigation and Rainfall on the Population Dynamics of Rift Valley Fever and Other Arbovirus Mosquito Vectors in the Epidemic-Prone Tana River County, Kenya.

Rift Valley fever (RVF) is a mosquito-borne viral zoonosis that is found in most regions of sub-Saharan Africa, and it affects humans, livestock, and some wild ungulates. Outbreaks are precipitated by an abundance of mosquito vectors associated with heavy persistent rainfall with flooding. We determined the impact of flood-irrigation farming and the effect of environmental parameters on the ecology and densities of primary and secondary vectors of the RVF virus (RVFV) in an RVF-epidemic hotspot in the Tana River Basin, Kenya. Mosquito sampling was conducted in farms and villages (settlements) in an irrigated and a neighboring nonirrigated site (Murukani). Overall, a significantly higher number of mosquitoes were collected in farms in the irrigation scheme compared with villages in the same area (P < 0.001), or farms (P < 0.001), and villages (P = 0.03) in Murukani. In particular, key primary vectors of RVFV, Aedes mcintoshi Marks and Aedes ochraceous Theobald, were more prevalent in the farms compared with villages in the irrigation scheme (P = 0.001) both during the dry and the wet seasons. Similarly, there was a greater abundance of secondary vectors, particularly Culex univittatus Theobald and Culex pipiens (L.) in the irrigation scheme than in the Murukani area. Rainfall and humidity were positively correlated with mosquito densities, particularly the primary vectors. Adult floodwater mosquitoes and Mansonia spp. were collected indoors; immatures of Ae. mcintoshi and secondary vectors were collected in the irrigation drainage canals, whereas those of Ae. ochraceous and Aedes sudanensis Theobald were missing from these water bodies. In conclusion, irrigation in RVF endemic areas provides conducive resting and breeding conditions for vectors of RVFV and other endemic arboviruses.

The gametocyte-activating factor xanthurenic acid stimulates an increase in membrane-associated guanylyl cyclase activity in the human malaria parasite Plasmodium falciparum.

Sex is an obligate step in the life cycle of the malaria parasite and occurs in the midgut of the mosquito vector. With both Plasmodium falciparum and Plasmodium berghei, the tryptophan metabolite xanthurenic acid induces the release of motile male gametes from red blood cells (exflagellation), a prerequisite for fertilization. The addition of cGMP or phosphodiesterase inhibitors to cultures of mature gametocytes has also been shown to stimulate exflagellation. Here, we demonstrate that there is a guanylyl cyclase activity associated with mature P. falciparum gametocyte membrane preparations, which is dependent on the presence of Mg(2+)/Mn(2+) but is inhibited by Ca(2+). Significantly, this activity is increased on addition of xanthurenic acid. In contrast, a xanthurenic acid precursor (3-hydroxykynurenine), which is not an inducer of exflagellation, does not induce this guanylyl cyclase activity. These results therefore suggest that xanthurenic acid-induced exflagellation may be mediated by activation of the parasite cGMP signalling pathway.

Guanylyl cyclase activity associated with putative bifunctional integral membrane proteins in Plasmodium falciparum.

We report here that guanylyl cyclase activity is associated with two large integral membrane proteins (PfGCalpha and PfGCbeta) in the human malaria parasite Plasmodium falciparum. Unusually, the proteins appear to be bifunctional; their amino-terminal regions have strong similarity with P-type ATPases, and the sequence and structure of the carboxyl-terminal regions conform to that of G protein-dependent adenylyl cyclases, with two sets of six transmembrane sequences, each followed by a catalytic domain (C1 and C2). However, amino acids that are enzymatically important and present in the C2 domain of mammalian adenylyl cyclases are located in the C1 domain of the P. falciparum proteins and vice versa. In addition, certain key residues in these domains are more characteristic of guanylyl cyclases. Consistent with this, guanylyl cyclase activity was obtained following expression of the catalytic domains of PfGCbeta in Escherichia coli. In P. falciparum, expression of both genes was detectable in the sexual but not the asexual blood stages of the life cycle, and PfGCalpha was localized to the parasite/parasitophorous vacuole membrane region of gametocytes. The profound structural differences identified between mammalian and parasite guanylyl cyclases suggest that aspects of this signaling pathway may be mechanistically distinct.

Trypanosoma cruzi adenylyl cyclase is encoded by a complex multigene family.

The parasitic protozoan Trypanosoma cruzi undergoes several differentiation events during its life cycle. Some of these transitions are thought to involve activation of adenylyl cyclase via the binding of peptide ligands to the cell surface. Here we describe the characterisation of the adenylyl cyclase gene family of T. cruzi. Two complete genes and one pseudogene have been sequenced. The protein products appear to have a large extracellular domain, a single transmembrane helix and a cytosolic catalytic domain. The adenylyl cyclase genes are present on at least six chromosomes and are scattered rather than clustered. They form a large polymorphic family in which the extracellular domain is particularly variable. An Escherichia coli adenylyl cyclase mutant could be complemented by expression of the catalytic domain of the T. cruzi enzyme. The recombinant protein had adenylyl cyclase activity in vitro, which was enhanced by increasing concentrations of divalent cations (Mn2+ > Mg2+). This constitutively active recombinant protein will be a useful tool for dissecting the catalytic mechanism of adenylyl cyclase.

Chlorproguanil/dapsone for uncomplicated Plasmodium falciparum malaria in young children: pharmacokinetics and therapeutic range.

The disposition of chlorproguanil/dapsone (one daily dose for 3 d of 1.2 and 2.4 mg/kg respectively) has been studied in young children with Plasmodium falciparum malaria, to provide data complementary to a clinical trial of this drug combination. Unbound concentrations of chlorcycloguanil (the active metabolite of chlorproguanil) and dapsone in clinical samples have been related to the unbound drug concentrations which produced defined outcomes in tests in vitro of drug efficacy and toxicity. Twelve children with uncomplicated malaria were treated: all cleared parasitaemia within 72 h and made uneventful recoveries. After the first dose of chlorproguanil/dapsone the maximum unbound chlorcycloguanil concentration in clinical samples (19 ng/mL [about 60 nM]) was 2 orders of magnitude above the 50% inhibitory concentration (IC50) value for this drug against the K39 stain of P. falciparum, while falling 2 orders of magnitude below its IC50 against human bone marrow cells; the maximum unbound dapsone concentration in clinical samples (160 ng/mL [about 645 nM]) was 10-fold higher than its IC50 against the K39 strain. However, because of the rapid elimination of chlorproguanil from the body (half-life 12.6 +/- 6.3 h), the minimum fractional inhibitory concentrations of unbound chlorcycloguanil/dapsone against the K39 strain were probably exceeded for no more than 6 d. These data, together with the clinical trial, will be helpful in deciding whether current chlorproguanil/dapsone doses are optimal for the treatment of falciparum malaria.

The disposition of intramuscular artemether in children with cerebral malaria; a preliminary study.

The disposition of intramuscular artemether (AM) was studied in 26 Kenyan children with cerebral malaria. Antimalarial activity determined by bioassay was compared with total plasma AM plus dihydroartemisinin (DHA) determined by high power liquid chromatography (HPLC). Therapeutic levels were achieved in most subjects (21/26) within 1 h of receiving intramuscular AM (3.2 mg/kg), with close correlation between bioassay and HPLC measurements (r = 0.706). However, there was marked inter-individual variation, antimalarial activity was undetectable in 5 subjects ('non-absorbers'), and plasma concentrations were lower in subject with respiratory distress. The 50% parasite clearance time was significantly longer in non-absorbers (mean = 13.1 h, SD = 10.8 vs. mean = 7.8 h, SD = 5.5; P = 0.013). We conclude that the bioavailability of intramuscular AM in children with severe malaria may be highly variable, particularly in the presence of respiratory distress, and may be associated with an inadequate therapeutic response.

Measurement of physiological concentrations of dapsone and its monoacetyl metabolite: a miniaturised assay for liquid or filter paper-absorbed samples.

A modification of existing HPLC assay methods is described for the measurement of dapsone and monoacetyldapsone in 50-microliter samples of plasma and whole blood. This method, in particular the use of small sample volumes dried onto filter paper strips, is applicable to multi-sample clinical and pharmacokinetic studies in children with malaria, who are often anaemic, and where sample volume must be kept to a minimum. Basified samples were extracted into 5 ml of ethyl acetate-tert.-butylmethyl ether (1:1, v/v), chromatographed on a mu BondapaK C18, 10-micron column with water-acetonitrile-glacial acetic acid (81:17.5:5, v/v) containing 2 g/l l-octanesulphonic acid as the mobile phase and detected at 274 nm.

Cutaneous hypersensitivity reactions to thiacetazone, HIV infection and thiacetazone concentrations in plasma.

We have studied the relationship between the plasma concentration-time profile of thiacetazone over the 24 h between doses [AUC(0.24h)] and the incidence of cutaneous reactions among HIV-infected patients with tuberculosis in Kenya. Cutaneous reactions due to thiacetazone occurred in 4/14 [28.6%] HIV+ve patients compared with 3/47 [6.4%] HIV-ve patients [RR = 4.48, 95% CI-1.1 to 17.7], and all resolved on alternative therapy. Among the HIV+ve patients, those with cutaneous reactions had higher AUC(0.24h) values, although the difference was not significant. These results do not exclude pharmacokinetic change as being at least partly responsible for cutaneous reactions to TCZ in HIV+ve patients, and do not refute an immunological basis for the reaction. With regard to the operational use of TCZ in Africa, there is no indication that a modification of the dose will reduce the frequency of drug reactions.

Differential extraction of artemether and its metabolite dihydroartemisinin from plasma and determination by high-performance liquid chromatography.

A method is described for the separation of artemether (ARM) from its metabolite dihydroartemisinin (DHA) and determination by HPLC. The basis of the separation is differential extraction of the drugs from plasma as a function of plasma pH. Hexane extracted ARM from basiffied plasma and both ARM and DHA from normal plasma. Derivatized extracts were chromatographed on a 5-microns ODS column with water-acetonitrile (40:60) as mobile phase and detected at 254 nm. The method removes the need for expensive absorption cartridges (BondElut). Chromatography has been improved and the elution time shortened in comparison with previous methods.

Ferriprotoporphyrin catalysed decomposition of artemether: analytical and pharmacological implications.

The ability of the products of erythrocytic haemolysis and ferriprotoporphyrin (FP-IX)-containing substances of facilitate the decomposition of the antimalarial drug artemether has been evaluated in vitro. The products of haemolysis accelerate the degradation of artemether to unidentified compounds which are undetectable by currently available HPLC methods. This decomposition is temperature dependent and occurs after relatively brief artemether (ARM)-catalyst contact. Using radiolabelled [16-14C]ARM, we have demonstrated that the extractability of total radioactivity into the organic phase diminishes with increasing FP-IX concentration with an appreciable reduction in the organic extractability as ARM in the presence of FP-IX and associated increase in water solubility of radioactivity when the concentration of FP-IX increases from 0 to 7.7 mM. This effect appears to be temperature dependent for incubations with haematin. Characterisation of the organically extractable radioactivity from incubations of [16-14C]ARM with FP-IX has shown a loss of ARM and the formation of two radioactive products which occurs in proportion to the concentration of FP-IX. We believe these findings are of particular relevance to the determination of plasma concentrations of ARM and dihydroartemisinin (DHA) in malaria patients where extensive haemolysis and the presence of breakdown products of haemoglobin may contribute to a reduction in the circulating concentration of these substances. In these circumstances, measurement of ARM and DHA by conventional methods may be meaningless.

Measurement of halofantrine and its major metabolite desbutylhalofantrine in plasma and blood by high-performance liquid chromatography: a new methodology.

A new high-performance liquid chromatographic assay for the measurement of halofantrine and desbutylhalofantrine in plasma and whole blood is described. The method involves a smaller sample volume, simplified sample pre-treatment and a shorter run-time, and is adaptable to the measurement of samples dried onto filter paper strips. Using this method, which is both selective and sensitive, plasma concentration versus time profiles for both substances have been investigated following a single oral dose (500 mg) of halofantrine hydrochloride to a healthy adult volunteer. In addition, a clinical study designed to evaluate the disposition and elimination of the two compounds in children with non-severe falciparum malaria is in progress.