Knowledge and attitudes to malaria control and acceptability of permethrin impregnated sisal curtains.

OBJECTIVES: The objectives of this study were to conduct a survey on the knowledge and practices relating to malaria prevention and treatment in two rural communities in Western Kenya, and to determine the acceptability of specially designed permethrin impregnated sisal strands curtains previously introduced into one of the study communities as barriers to mosquito biting. DESIGN: A knowledge, attitudes and practices (KAP) survey was conducted by pre-trained interviewers using a pre-tested questionnaire. SETTING: The study was conducted in two communities located 15 km northwest of Kisumu town, and next to the swamps bordering Lake Victoria in Kisumu District, Western Kenya. PARTICIPANTS: Adult individuals from 50 houses selected from the intervention, and 50 houses from the control sites were included in the study. RESULTS: Both communities had a clear conception of malaria and its symptoms, and of the mosquito as its vector. Malaria was recognised as a potential cause of death by 44% and 72% of the participants in the intervention and control sites respectively. Sixty two per cent to 74% of the people interviewed in both sites named mosquitoes as the causative agents of malaria. Chloroquine was still the drug of choice for malaria treatment, with over 70% usage among the study population. Mention of pyrimethamine/sulfadoxine and pyrimethamine/sulfalene as alternative therapy was below five per cent. Despite a high level (86%) of awareness of bednets as effective barriers to mosquito biting, they were reported in use by less than 35% in both communities. Sisal strand curtains were considered effective and acceptable to more than 80% of the community. CONCLUSIONS: The results of this study underscore the existing need for affordable means of mosquito control, such as sisal strand curtains, for such rural communities which may be acutely aware of the problems associated with malaria, but are constrained from taking any action by lack of resources.

Plasmodium falciparum circumsporozoite vaccine immunogenicity and efficacy trial with natural challenge quantitation in an area of endemic human malaria of Kenya.

It has been hypothesized that antibody induced by Plasmodium falciparum circumsporozoite protein vaccine would be effective against endemic human malaria. In a malaria endemic region of Kenya, 76 volunteers, in 38 pairs sleeping adjacently, were immunized with subunit circumsporozoite protein Asn-Ala-Asn-Pro tetrapeptide repeat-pseudomonas toxin A, or hepatitis B vaccine. After quinine and doxcycycline, volunteers were followed for illness daily, parasitemia weekly, antibody, T-lymphocyte responses, and treated if indicated. Anopheles mosquitoes resting in houses were collected, and tested for P. falciparum antigen, or dissected for sporozoites and tested for blood meal ABO type and P. falciparum antigen. Vaccine was safe, with side-effects similar in both groups, and immunogenic, engendering IgG antibody as high as 600 micrograms ml-1, but did not increase the proportion of volunteers with T-lymphocyte responses. Estimation of P. falciparum challenge averaged 0.194 potentially infective Anopheles bites/volunteer/ day. Mosquito blood meals showed no difference in biting intensity between vaccine and control groups. Both groups had similar malaria-free survival curves, cumulative positive blood slides, cumulative parasites mm-3, and numbers of parasites mm-3 on first positive blood slide, during three post-vaccination observation periods. Every volunteer had P. falciparum parastemia at least once. Vaccinees had 82% and controls 89% incidences of symptomatic parasitemia (P = 0.514, efficacy 9%, statistical power 95% probability of efficacy < 50%). Vaccine-induced anti-sporozoite antibody was not protective in this study. Within designed statistical precisions the present study is in agreement with efficacy studies in Colombia, Venezuela and Tanzania.

Screening of metal ion chelators against Leishmania donovani-infected Syrian hamsters

Leishmania donovani-infected Syrian hamsters were treated intraperitoneally with 0.23 mmoles/kg/day of EDTA; EGTA; HEEDTA and 100 mg/kg/day of Pentostam R. The control group received 0.1 ml of phosphate buffered saline. After 30 days of treatment; the animals were sacrificed. Of the Pentostam-treated animals; 5 out 6 had negative spleen cultures; while all the chelator and PBS-treated ones yielded parasites. While all the Pentostam-treated hamsters yielded had negative bone marrow cultures; only 1 out of 6 HEEDTA-treated hamsters yielded parasites. Spleen; liver and bone marrow parasite-loads calculated from chelator-treated animals were consistently significantly higher than for Pentostam-treated animals. These results suggest that although metal ion chelators have some antileishmanial potential; their in vivo activity against L. donovani is low compared to Pentostam

Comparison of Giemsa and acridine orange stains for the diagnosis of Leishmania donovani in biopsies of infected hamsters

Identical impression smears of spleen; liver and bone marrow biopsy materials from Leishmania donovani-infected hamsters were stained using either acridine orange or Giemsa. Spleen parasite-loads calculated from the two stains for identical biopsy material were significantly different from each other. However; liver and bone marrow parasite-loads calculated from either Giemsa-stained or acridine orange-stained biopsies were not significantly different from each other. This study has shown that acridine orange; which is a quick and simple technique; has great potential in the diagnosis of kala-azar when liver and bone marrow biopsies are used

Determination of the optimal EDTA and Pentostam concentration in the treatment of Leishmania donovani-infected laboratory animal rodent models

The objective of this study was to undertake a dose response study to determine the optimal Pentostam and Ethylenediamine tetraacetic acid (EDTA) dose that could be used in the treatment of leishmania-infected golden hamsters or BALB/c mice for a period of 30 days. This pilot experiment was done using only one chelator; EDTA and the toxicity results obtained from this experiment formed the basis for the selection of a suitable chelator dose of this class for the future treatment of leishmania-infected laboratory animal rodent models. It is concluded that Pentostam concentrations beyond 600 mg/kg are highly toxic to mice and therefore unsuitable for use. Although Pentostam have been used to treat leishmania-infected BALB/c mice; this study has shown that a concentration of 100 mg/KG/day is the most suitable dose for use in the treatment of rodent animal models

Screening of metal ion chelators against Leishmania donovani-infected BALB/c mice

Previous in vitro experiments by Mbati et al. have shown that Ethylenediamine tetraacic acid (EDTA) and Ethyleneglycol-bis (B-aminoethyl ether) N;N;N1;N1; tetraacetic acid (EGTA) substantially reduce parasite burdens of leishmania donovani in either cell free media or when engulfed in mouse peritoneal macrophages. The objective of this study was to compare the activity of the same chelators against Leishmania donovani in BALB/c mice infected with a much lower parasite inoculum

In vitro experimental antileishmanial potential of ethylenediamine tetraacetic acid, disodium salt.

Leishmania donovani promastigotes (NLB 065) were treated to various concentrations of ethylenediamine tetraacetic acid (EDTA), disodium salt for 7 days. Concentrations of up to 1.0 mg/ml produced no significant reduction in the population of Leishmania promastigotes cultured in RPMI 1640 medium supplemented with 15% foetal calf serum and incubated at 27 degrees C (P greater than 0.05). Concentrations between 0.05 and 0.1 mg/ml EDTA did not significantly affect the viability of Balb/c mouse peritoneal macrophages (P greater than 0.05) whereas those equal to or above 0.2 mg/ml were severely toxic to macrophages (P less than 0.05; n = 8). Leishmania parasites engulfed in mouse peritoneal macrophages were treated to various concentrations of ethylenediamine tetraacetic acid (EDTA) disodium salt. The higher the EDTA concentration within the acceptable toxic levels for macrophages (less than or equal to 0.1 mg/ml), the higher was the rate at which parasites were cleared (p less than 0.05). The length of time in days within which infected macrophages were treated contributed significantly to a decline in the level of parasite-loads (P less than 0.05; n = 10). Analysis of mean metal ion values in mg/1 using atomic absorption spectrometry revealed that the levels of calcium, magnesium, and iron were virtually unchanged in the supernatant of variant culture experiments (P greater than 0.05). The level of manganese was higher in ethylenediaiene tetraacetic acid (EDTA), disodium salt-treated cultures as compared to the control (P less than 0.05; n = 20).(ABSTRACT TRUNCATED AT 250 WORDS)

An experimental model system for leishmaniasis. An ultrastructural study on cultured macrophages exposed to Leishmania parasites and sodium stibogluconate.

To facilitate studies on the effect of chemotherapeutic agents on the host-parasite interaction in leishmaniasis, we have developed an experimental model for infecting mouse peritoneal macrophages in culture with recently-isolated Leishmania donovani promastigotes. As the drug action is often dependent on concentration, the distribution of sodium stibogluconate, which is the commonly used drug for treatment of leishmaniasis, was studied in various parts of the macrophages by energy dispersive X-ray microanalysis. The drug was found to accumulate in secondary lysosomes. The ultrastructural examination, using TEM and SEM, of macrophages, whose secondary lysosomes had been preloaded with gold particles, showed that leishmania parasites are phagocytosed and finally located in secondary lysosomes. Using flameless atomic absorption spectrophotometry, the concentration of Mn, Fe and Cu in promastigotes of Leishmania donovani, Leishmania aethiopica, Leishmania crithidia, Leishmania major and their culture media was estimated. Of the three transition metals, the parasites accumulated only Mn from the medium, which they may use in a primitive defense mechanism against reactive oxygen metabolites produced by macrophages during the respiratory burst associated with phagocytosis.

An experimental model system for leishmaniasis. Effects of porphyrin-compounds and menadione on Leishmania parasites engulfed by cultured macrophages.

In order to facilitate studies on the effects of chemotherapeutic agents on the host-parasite interactions in leishmaniasis, we have developed an experimental model for infecting human monocyte-derived- and mouse peritoneal macrophages in culture with recently-isolated Leishmania donovani promastigots (LDP). The chemotherapeutic agents studied were protoporphyrin, hematoporphyrin, menadione, and combinations of hematoporphyrin plus menadione. Since the Leishmania donovani amastigotes survived poorly in mouse macrophages and protoporphyrin was quite toxic to the latter, our investigations were focused on the effects of hematoporphyrin and menadione on amastigotes engulfed by human macrophages. Treatment of Leishmania donovani amastigotes-infested human macrophages with either 50 microM hematoporphyrin or 10 microM menadione did not influence significantly the survival of either Leishmania donovani amastigotes or the macrophages themselves. Larger individual doses of hematoporphyrin and menadione were toxic to both parasites and macrophages. The combination of 50 microM hematoporphyrin and 10 microM menadione, however, caused the destruction of the parasites without affecting the host macrophage. The enhanced deleterious effect from combined low doses of hematoporphyrin and menadione is discussed in terms of the production of reactive oxygen species, such as superoxide anion radical and hydrogen peroxide, originating from cellular redox cycling of menadione, and followed by decomposition of the formed hydrogen peroxide by hematoporphyrin to produce the more reactive hydroxyl radical.

Effects of ligation and irradiation on the submaxillary glands in rats.

Rat submaxillary glands were exposed to excretory duct ligation, high-energy electron irradiation, or a combination of both. Ligation was done on the left excretory ducts, while 24 Gy irradiation was given within 8 days in three divided doses of 8 Gy. In the combined insults, the excretory ducts (one duct per rat) were first ligated, followed by 24 Gy irradiation. The effects were analysed by morphological methods. Ligation of excretory ducts caused morphological disorganization of the gland parenchyma, with resultant disappearance of secretory granules of serous cells, shrinkage of mucous cells, dilation of ducts, pronounced fibrosis and infiltration of inflammatory cells. When irradiated, some serous cells disappeared, leaving empty fibrosed spaces, while mucous cells showed no consequential radio-damage. The combined treatment of ligation of excretory ducts and irradiation caused extensive morphologic evidence of irreversible damage, i.e. no serous cells remained, disfigured and very shrunk mucous cells extremely dilated ducts, infiltration of leukocytes and fibrotic changes of the glands.

A histological study of the effects of different alpha-adrenergic and cholinergic agonists on the rat submandibular gland.

Rat submandibular glands were exposed to various sialagogues, including carbachol, clonidine, noradrenaline and cyclocytidine. The effects of these drugs were morphologically compared. Clonidine, which is an alpha-2-agonist, caused no depletion of granules in the serous cells examined. Noradrenaline and cyclocytidine, which are alpha-1-agonists, showed remarkable depletion of secretory granules in the serous cells. Carbachol caused visible and abundant salivation in the animals, but was found to produce only partial granular depletion of both serous and mucous cells. To induce experimentally a complete depletion of granular serous cells, cyclocytidine was found to be an excellent choice as a sialagogue with no side-effects on the cardiac and respiratory system.

Radiation effects on mast cells: secretory ability, histamine release and recovery, and cell number.

We studied the effects of irradiation with a single fraction of high-energy electrons, in the absorbed dose range used in radiotherapy, on features related to the rat mesenteric mast-cell (MC) population. Such features were energy dispersive X-ray microanalysis for zinc, light and transmission electron microscopic changes, histamine content, and secretory ability of MCs when exposed to the potent secretagogue 48/80. The observation period was 14 days. Also studied were the effects of radiation on the number and histamine content of free peritoneal MCs. Irradiation-induced decrease in histamine content was delayed for at least two days and the rate of subsequent recovery of mesenteric histamine was approximately 70% below normal. This was corroborated by the findings of necrotic and fragmented mesenteric MCs during the entire experimental period. Fibroblasts and macrophages phagocytosed groups of MC granules as well as individual MC granules. A fraction of the extruded MC granules retained an almost normal appearance. This suggests that they were solubilized unduly slow presumably because the irradiation destroyed the mechanisms and enzymes which normally degrade the heparin-protein granule matrix and its heparin. Moreover, the irradiation suppressed the secretory ability of mesenteric MCs and significantly reduced the number of free peritoneal MCs. The findings indicate that the MC is a remarkably radiosensitive connective-tissue cell.

Dimethylsulfoxide increases the survival and lysosomal stability of mouse peritoneal macrophages exposed to low-LET ionizing radiation and/or ionic iron in culture.

Reactive oxygen-derived free radicals form excessively during irradiation of biological structures, but also normally in many cellular oxidative processes, albeit in small amounts. Unless scavenged by protective mechanisms, such radicals may induce peroxidation of polyunsaturated fatty acids resulting in membrane damage. The process may be catalysed to a considerable extent by transitional metals with the capacity to form redox systems, such as Fe3+ in equilibrium Fe2+. In the present study, it is shown that radiation by X-rays and/or exposure to ionic iron (Fe3+) causes decreased survival in parallel with lysosomal labilization of cultured mouse peritoneal macrophages (MPMs). The latter event was demonstrated as a reduced capacity of lysosomes in living MPMs to retain acridine orange during photo-oxidative stress caused by continuous exposure to blue light of short wavelength. The effects of X-irradiation, and/or lysosomal iron-loading, could be counteracted by the addition of the .OH-scavenging drug dimethylsulfoxide (DMSO) to the cell culture medium. The findings suggest that X-irradiation may damage certain sensitive G0 cells, such as Kupffer cells, serous cells of salivary glands and old macrophages, which normally have substantial concentrations of metals within their vacuolar apparatus, possibly by lysosomal damage involving .OH-mediated lipid peroxidation.

Morphologic and histochemical studies on the differing radiosensitivity of ductular and acinar cells of the rat submandibular gland.

The submandibular glands of male rats were exposed to 50 Gy X-irradiation as a single dose, with or without pre-treatment with either alpha-adrenergic agonists (noradrenaline, phenylephrine) or a cholinergic antagonist (atropine). The effects were analyzed by morphometric, cytochemical and biophysical methods. When X-irradiated without drug pre-treatment, many serous epithelial cells of the intralobular convoluted ducts displayed morphologic evidence of irreversible radiation damage, in contrast to neighbouring mucous and other cells which were unaffected. The effect was maximal 96 h after irradiation. Serous cells from animals irradiated after pre-treatment with atropine showed much more wide-spread injury than those of animals exposed to X-irradiation only. In contrast , serous cells suffered considerably less damage if their secretory granules had been depleted 1 or 2 h before irradiation with either noradrenaline or phenylephrine. Other epithelial cells showed no modulation of their slight radioresponsiveness by these drugs. The observations were substantiated by morphometry of three cell types: (a) mucous cells, (b) non-granulated serous and intralobular striated duct cells, and (c) granulated serous cells. The findings suggest that the striking radiosensitivity of salivary gland serous epithelial cells is linked to their content of secretory granules. These granules are rich in heavy metals, as demonstrated cytochemically with the sulphide silver method (SSM). Using particle-induced X-ray emission ( PIXE ) spectroscopy, the principal metals were shown to be Zn, Mn and Fe. It is conceivable that membranes which enclose organelles rich in metals with the ability to form redox systems (e.g. Fe2+ in equilibrium Fe3+) show enhanced sensitivity to radiation damage due to the metal-catalyzed induction of lipid peroxidation by ionizing radiation. Disruption of secretory granules would be expected to release lytic enzymes into the cell sap, resulting in autolysis. This hypothesis is supported by the findings that atropine--which increases the number of granulated serous cells--enhances radiosensitivity, while noradrenaline and phenylephrine--which cause degranulation of serous cells--decrease radiosensitivity.

Effect of iron on the stability of macrophage lysosomes.

Mouse peritoneal macrophages exposed to 60 microM Fe3+ in culture showed a pronounced decline in survival rate and an enhanced capacity for lipid peroxidation, as estimated by the formation of malondialdehyde. The iron-exposed cells contain secondary lysosomes loaded with iron as demonstrated by transmission electron microscopy, energy dispersive X-ray micro-analysis and the sulphide-silver method. The macrophages concentrated within their lysosomes the weak base acridine orange (AO) when the latter was added to the medium in a low concentration (1:10(5)). When cells were viewed under continuous illumination with short wave blue light in a fluorescence microscope, AO-exposed cells initially showed a brilliant red granular fluorescence and subsequently a progressive redistribution of AO into the cell sap and the nucleus with resultant green fluorescence. This redistribution of AO was associated with the disappearance of the normal granular reaction product pattern of the lysosomal marker enzyme acid phosphatase (AP), as demonstrated with a Gomori-type reaction, and later with cell death. The redistribution of AO and AP from the lysosomal vacuome to the cell sap under the influence of blue light occurred markedly more rapidly in macrophages containing iron loaded lysosomes than in control cells. The findings indicate that the presence of iron in lysosomes decreases the proton-retaining ability of their bordering membranes, possibly by way of enhanced metal (iron) catalyzed lipid peroxidation. It appears that leakage of AO signifies destabilization of the lysosomal membrane.

Macrophage radiosensitivity in culture as a function of exposure to ionic iron.

The effects of various concentrations of Fe3+ (30-125 microM) and various doses of X-irradiation (10 to 30 Gy) on the survival of mouse peritoneal macrophages in culture was tested and compared with the effect of combinations of both Fe3+ and X-irradiation. Exposure of the cells to 30 microM Fe3+ or 10 Gy did not significantly alter the survival pattern, as compared with control cells. Greater dosages of iron or X-irradiation caused increasing cellular degeneration and death. When the combined effect of 30 microM Fe3+ and 10 Gy was tested a pronounced decrease in survival time was noted. The production of malondialdehyde (MDA) was studied in cells cultured with and without iron and after exposure to X-irradiation. The results indicated an increasing peroxidative capacity in parallel with increasing dose and extent of exposure to iron and irradiation. Combinations of 30 microM Fe3+ and 10 Gy resulted in enhanced MDA production as compared with these treatments alone (in which levels of MDA did not differ from those in controls). The findings support the theory that cells containing iron-loaded lysosomes are abnormally sensitive to X-irradiation due to the catalytic activity of the Fe2+ in equilibrium with Fe3+ redox system on lipid peroxidation. The postulated mechanism involves disruption of lysosomal membranes as a consequence of increased lipid peroxidation, resulting in leakage of potent lytic enzymes into the cell sap causing cellular degeneration and death.