A murine malaria protocol for characterizing transmission blocking benefits of antimalarial drug combinations

Background. Current efforts towards malaria elimination include the discovery of new transmission blocking (TB) drugs and identification of compounds suitable to replace primaquine, recommended as transmission blocking post treatment after artemisinin combination therapy (ACT). High through put screening of compound libraries has allowed to identify numerous compounds active in vitro against gametocytes and insect early sporogonic stages, but few studies have been performed to characterize TB compounds in vivo. Here we propose a double TB drug Direct Feeding Assay (2TB-DFA), suitable to assess the combined effects of TB compounds. Materials and methods. Plasmodium berghei GFPcon (PbGFPcon), BALB/c mice and Anopheles stephensi mosquitoes were used. Artemisinin (ART) and artesunate (AS) served as examples of artemisinins, NeemAzal® (NA), as a known TB-product with sporontocidal activity. DFA experiments were performed to assess the appropriate time point of administration before mosquito feeding and estimate suitable sub-optimal doses of the three compounds that allow combination effects to be appreciated. Results. Suboptimal dosages, that reduce about 50% of oocyst development, were recorded with ART in the range of 16-30 mg/kg, AS 14-28 mg/kg and NA 31-38mg/kg. Ten hours before mosquito feeding (corresponding to 3.5 days after mouse infection) was determined as a suitable time point for mouse treatment with ART and AS and 1 hour for post-treatment with NA. ART given at 35 mg/kg in combination with NA at 40 mg/kg reduced oocyst density by 94% and prevalence of infection by 59%. Similarly, the combination of ART at 25 mg/kg plus NA at 35 mg/kg decreased oocyst density by 95% and prevalence of infection by 34%. In the 2TB-DFA, conducted with AS (20 mg/kg) and NA (35 mg/kg) the combination treatment reduced oocyst density by 71% and did not affect prevalence of infection. Applying ‘Highest Single Agent’ analysis and considering as readout oocyst density and prevalence of infection, cooperative effects of the combination treatments, compared with the single compound treatments emerged. Conclusion. This study suggests the 2TB-DFA to be suitable for the profiling of new TB candidates that could substitute primaquine as a post-treatment to ACT courses

Exploring communities’ and health workers’ perceptions of indicators and drivers of malaria decline in Malindi, Kenya

Background. Since 2000, a decrease in malaria burden has been observed in most endemic countries. Declining infection rates ad disease burden and reduction in asymptomatic carriers are the outcome of improved quality of care and relaled health system factors. These include improved case management through better diagnosis, implementation of highly effective antimalarial drugs and increased use of bednets. We studied communites' and health workers' perceptions of indicators and drivers in the context of decreasing malaria transmission in Malindi, Kenya. Providing residents with bednets contributed to malaria reduction and increasing community awareness on the causes and symptoms of malaria and imroved malara treatment were also perceived to contribute to the decline of malaria. The study identified three perceived drivers to the reported decline in malaria: a) community health workers' enhanced awareness creation towards household owners regarding malaria-related activities through visitations and awarenss sessions, b) women involvement in Savings Internal Lending Community was perceived to have increased their financial base, thereby improving their decision-making power toward the care of their sick child(ren), c) non governmental Organisations (NGOs) and partners played a promoter part in health and general economic development initiatives.. Conclusion: to achieve the goal of malaria elimination, collaboration beween governmental and NGOs will be crucial when improving the financial base of women and enhancing participation of community health workers

Detecting malaria sporozoites in live, field-collected mosquitoes

A method is described for identifying malaria-infected mosquitoes, without killing them or hampering their fitness. Individual mosquitoes were induced to salivate on coverslips, and sporozoites, deposited on the glass surface, were visualized by Giemsa staining. Of 21 mosquitoes found to contain sporozoites by salivary gland dissection, 13 had delivered sporozoites on coverslips. A positive correlation was found between the amount of saliva expelled and ejection of sporozoites, indicating that the sensitivity of the method may be increased by improving the probing behaviour of the mosquitoes. The procedure described may be suitable for selecting infected mosquitoes which are able to eject sporozoites during probing. Being applicable to wild Anopheles and to large numbers of mosquitoes, the method lends itself for use in field studies on malari

Detecting malaria sporozoites in live, field-collected mosquitoes

A method is described for identifying malaria-infected mosquitoes, without killing them or hampering their fitness. Individual mosquitoes were induced to salivate on coverslips, and sporozoites, deposited on the glass surface, were visualized by Giemsa staining. Of 21 mosquitoes found to contain sporozoites by salivary gland dissection, 13 had delivered sporozoites on coverslips. A positive correlation was found between the amount of saliva expelled and ejection of sporozoites, indicating that the sensitivity of the method may be increased by improving the probing behaviour of the mosquitoes. The procedure described may be suitable for selecting infected mosquitoes which are able to eject sporozoites during probing. Being applicable to wild Anopheles and to large numbers of mosquitoes, the method lends itself for use in field studies on malari

Insecticide-treated curtains reduce the prevalence and intensity of malaria infection in Burkina Faso

A large, randomized controlled trial to investigate the impact of insecticide-treated curtains (ITC) on child mortality was conducted in an area of seasonal, holoendemic malaria in Burkina Faso. 158 communities totalling some 90,000 people were censused and grouped into 16 geographical clusters, 8 of which were randomly selected to receive ITC in June-July 1994, just prior to the rainy season. In September-October 1995, at the peak period of malaria transmission, a cross-sectional survey was conducted in 84 of the villages. A random sample of 905 children aged 6-59 months was identified and visited. 763 children (84%) were present at the time of the visit and recruited into the study. Mothers were asked about fever in the past 24 h, the child's temperature was taken, and a sample of blood collected to identify and quantify malaria infections and to measure haemoglobin (Hb) levels. Children protected by ITC were less likely to be infected with Plasmodium falciparum (risk ratio = 0.92; 95% CI 0.86, 0.98) or P. malariae (risk ratio = 0.42, 95% CI 0.19, 0.95). The mean intensity of P. falciparum infections was lower among children protected by ITC (899 vs. 1583 trophozoites/microliter; P < 0.001), while the mean Hb level was 0.4 g/dl higher (P < 0.001). While we found no evidence that ITC had an impact on the prevalence of malaria-associated fever episodes, the confidence intervals around our estimates of the impact of ITC on malaria morbidity were wide. We conclude that widespread implementation of ITC in this area of high malaria transmission led to a modest reduction in the prevalence of malaria infection and to a more substantial reduction in the intensity of these infections which caused increased Hb levels. We were unable to demonstrate any impact of ITC on malaria morbidity, but the wide confidence intervals around our point estimates do not preclude the possibility of a substantial impact

In vitro multistage malaria transmission blocking activity of selected malaria box compounds

Purpose: Continuous efforts into the discovery and development of new antimalarials are required to face the emerging resistance of the parasite to available treatments. Thus, new effective drugs, ideally able to inhibit the Plasmodium life-cycle stages that cause the disease as well as those responsible for its transmission, are needed. Eight compounds from the Medicines for Malaria Venture (MMV) Malaria Box, potentially interfering with the parasite polyamine biosynthesis were selected and assessed in vitro for activity against malaria transmissible stages, namely mature gametocytes and early sporogonic stages. Methods: Compound activity against asexual blood stages of chloroquine-sensitive 3D7 and chloroquine-resistant W2 strains of Plasmodium falciparum was tested measuring the parasite lactate dehydrogenase activity. The gametocytocidal effect was determined against the P. falciparum 3D7elo1-pfs16-CBG99 strain with a luminescent method. The murine P. berghei CTRP.GFP strain was employed to assess compounds activities against early sporogonic stage development in an in vitro assay simulating mosquito midgut conditions. Results: Among the eight tested molecules, MMV000642, MMV000662 and MMV006429, containing a 1,2,3,4-tetrahydroisoquinoline-4-carboxamide chemical skeleton substituted at N-2, C-3 and C-4, displayed multi-stage activity. Activity against asexual blood stages of both strains was confirmed with values of IC50 (50% inhibitory concentration) in the range of 0.07\u20130.13 \ub5M. They were also active against mature stage V gametocytes with IC50 values below 5 \ub5M (range: 3.43\u20134.42 \ub5M). These molecules exhibited moderate effects on early sporogonic stage development, displaying IC50 values between 20 and 40 \ub5M. Conclusion: Given the multi-stage, transmission-blocking profiles of MMV000642, MMV000662, MMV006429, and their chemical characteristics, these compounds can be considered worthy for further optimisation toward a TCP5 or TCP6 target product profile proposed by MMV for transmission-blocking antimalarials

Effetti economici e sociali del sisma sugli allevamenti dell’Alto Maceratese

Il contributo analizza la filiera zootecnica dell’Alto Maceratese danneggiata dal sisma del 2016. Rispetto ad alcune difficolta storiche (marginalità territoriale e spopolamento), questa analisi si è concentrata sugli effetti del sisma. In collaborazione con i veterinari della Regione Marche, è stato somministrato ad un campione di 55 famiglie di allevatori un questionario semi-strutturato al fine di osservare le caratteristiche socio-economiche degli allevamenti (prevalentemente a media quota e dediti nella metà dei casi unicamente all’attività zootecnica) e i danni prodotti dal sisma (ad abitazioni, patrimonio zootecnico e strutture agricole). L’ultima sezione del questionario raccoglie alcune proposte di miglioramento nella fase di ricostruzione. Proprio la rigenerazione dei territori colpiti, infatti, sembra porre ambizione sfide che riguardano il ripristino delle attività lavorative e il ritorno della popolazione sul territorio

The Changing Epidemiology of Malaria in Ifakara Town, Southern Tanzania.

Between 1995 and 2000 there were marked changes in the epidemiology of malaria in Ifakara, southern Tanzania. We documented these changes using parasitological and clinical data from a series of community- and hospital-based studies involving children up to the age of 5 years. There was a right shift and lowering in the age-specific parasite prevalence in the community-based cohort studies. The incidence of clinical malaria in placebo-receiving infants in additional study cohorts dropped from 0.8 in 1995 to 0.43 episodes per infant per year in 2000, an incidence rate ratio of 0.53 (95% confidence interval: 0.404, 0.70, P<0.0001). At the same time, there was an increase in the total number of malaria admissions and a marked right shift in the age pattern of these admissions (median age in 1995 1.55 years vs. 2.33 in 2000, P<0.0001). However, the burden of malaria deaths remained in infants. We discuss how these dramatic changes in the epidemiology of malaria may have arisen from the use of currently available malaria control tools. Caution is required in the interpretation of hospital-based data as it is likely to underestimate the impact of anaemia on mortality in the community, where most paediatric deaths occur. Even in low/moderate malaria transmission settings, where older children suffer most malaria episodes, targeting effective malaria control at infants may produce important reductions in infant mortality caused by malaria

In vitro multistage malaria transmission blocking activity of selected Malaria Box compounds

Purpose: Continuous efforts into the discovery and development of new antimalarials are required to face the emerging resistance of the parasite to available treatments. Thus, new effective drugs, ideally able to inhibit the Plasmodium life-cycle stages that cause the disease as well as those responsible for its transmission, are needed. Eight compounds from the Medicines for Malaria Venture (MMV) Malaria Box, potentially interfering with the parasite polyamine biosynthesis were selected and assessed in vitro for activity against malaria transmissible stages, namely mature gametocytes and early sporogonic stages. Methods: Compound activity against asexual blood stages of chloroquine-sensitive 3D7 and chloroquine-resistant W2 strains of Plasmodium falciparum was tested measuring the parasite lactate dehydrogenase activity. The gametocytocidal effect was determined against the P. falciparum 3D7elo1-pfs16-CBG99 strain with a luminescent method. The murine P. berghei CTRP.GFP strain was employed to assess compounds activities against early sporogonic stage development in an in vitro assay simulating mosquito midgut conditions. Results: Among the eight tested molecules, MMV000642, MMV000662 and MMV006429, containing a 1,2,3,4-tetrahydroisoquinoline-4-carboxamide chemical skeleton substituted at N-2, C-3 and C-4, displayed multi-stage activity. Activity against asexual blood stages of both strains was confirmed with values of IC50 (50% inhibitory concentration) in the range of 0.07-0.13 µM. They were also active against mature stage V gametocytes with IC50 values below 5 µM (range: 3.43-4.42 µM). These molecules exhibited moderate effects on early sporogonic stage development, displaying IC50 values between 20 and 40 µM. Conclusion: Given the multi-stage, transmission-blocking profiles of MMV000642, MMV000662, MMV006429, and their chemical characteristics, these compounds can be considered worthy for further optimisation toward a TCP5 or TCP6 target product profile proposed by MMV for transmission-blocking antimalarials

Effects of Azadirachta indica seed kernel extracts on early erythrocytic schizogony of Plasmodium berghei and pro-inflammatory response in inbred mice

Background: Medicinal plant research may contribute to develop new pharmacological control tools for vector borne diseases, such as malaria. Methods: The effects of methanol extracts (ME) obtained from seed kernel of ripe and unripe Azadirachta indica fruits were studied on erythrocytic proliferation of the rodent malaria parasite Plasmodium berghei strain ANKA and on mice pro-inflammatory response, as evaluated by measuring the matrix-metalloproteinase-9 (MMP-9) and tumour necrosis factor (TNF) plasma levels, in two mouse strains (C57BL/6 and BALB/c) which are considered as prototypical of Th1 and Th2 immune response, respectively. Results: ME obtained from seed kernel of unripe Azadirachta indica fruits decreased by about 30% the proportion of erythrocytes infected with the malaria parasite in C57BL/6 mice in the 4 days suppressive test. In this treatment group, MMP-9 and TNF levels were notably higher than those measured in the same mouse strain treated with the anti-malarial drug artesunate, Azadirachta indica kernel extracts from ripe fruits or solvent. In BALB/c mice, treatment with kernel extracts did not influence parasitaemia. MMP-9 and TNF levels measured in this mouse strain were notably lower than those recorded in C57BL/6 mice and did not vary among treatment groups. Conclusions: The effects of the ME on the parasite-host interactions appeared to be mouse strain-dependent, but also related to the ripening stage of the neem fruits, as only the unripe fruit seed kernel extracts displayed appreciable bioactivity