In-vitro Activity of Artemether, Lumefantrine, Dihydroartemisinin and Piperaquine against 3D7 and W2 Plasmodium Falciparum Reference Clones in a Formulated Transport Medium and EDTA

Introduction: in vitro drug sensitivity relies on the growth of plasmodium in the presence of the antimalarials. These fresh isolates brought in for assay from collection sites normally not near the laboratory require a medium to keep the parasites viable in order to produce results which are a representation of the field situation in terms of drug susceptibility. However, factors  such  as  mixed  infection  of  drug resistant and  sensitive  parasites occurring in most field isolates can  influence  drug  test  outcome because they have different drug susceptibilities, thus there is a need of using clones which have identified these drug resistant sub populations to prevent treatment failures, misidentification of a populations susceptibility profile and also in any evaluation procedure so as to provide accurate test results. Objective: This study validated the use of a formulated transport medium (TM) by assaying clones which have been culture adapted in the lab for 50% inhibitory concentrations (IC50) against four antimalarials using SYBR Green 1 in vitro assay. Method: These clones (3D7 and W2) were revived and culture adapted for assay. Prior to subjecting them to drugs, they were exposed to 2ml formulated TM and the conventional EDTA anticoagulant in 5ml vacutainer tubes to act as control. Results: Both clones gave successful assays with moderate RFU values of between 2000 to 4000 parasites obtained by 3D7clone while W2 had RFU values as high as 20,000 parasites. However, their curves converged into discernible dose-response curves. Similarity was detected with no significant difference (P >0.05) observed in the medians inhibitory concentration 50 (IC50) between samples in the EDTA anticoagulant and TM however, samples in EDTA generally had higher IC50 values. Conclusion: This study concludes that the formulated TM holds promise for future use.

Water Hyacinth Eichhornia crassipes (Mart.) Solms-Laubach Dynamics and Succession in the Nyanza Gulf of Lake Victoria (East Africa): Implications for Water Quality and Biodiversity Conservation

This study, conducted in Nyanza Gulf of Lake Victoria, assessed ecological succession and dynamic status of water hyacinth. Results show that water hyacinth is the genesis of macrophyte succession. On establishment, water hyacinth mats are first invaded by native emergent macrophytes, Ipomoea aquatica Forsk., and Enydra fluctuans Lour., during early stages of succession. This is followed by hippo grass Vossia cuspidata (Roxb.) Griff. in mid- and late stages whose population peaks during climax stages of succession with concomitant decrease in water hyacinth biomass. Hippo grass depends on water hyacinth for buoyancy, anchorage, and nutrients. The study concludes that macrophyte succession alters aquatic biodiversity and that, since water hyacinth infestation and attendant succession are a symptom of broader watershed management and pollution problems, aquatic macrophyte control should include reduction of nutrient loads and implementing multifaceted approach that incorporates biological agents, mechanical/manual control with utilization of harvested weed for cottage industry by local communities

Presence and distribution of mosquito larvae predators and factors influencing their abundance along the Mara River, Kenya and Tanzania

Among all the malaria controlling measures, biological control of mosquito larvae may be the cheapest and easiest to implement. This study investigated baseline predation of immature mosquitoes by macroinvertebrate predators along the Mara River, determined the diversity of predators and mosquito larvae habitats and the range of their adaptive capacity to water physico-chemical parameters. Between July and August 2011, sampling sites (n=39) along the Mara River were selected and investigated for the presence of macroinvertebrate predators and mosquito larvae. The selected sampling sites were geocoded and each dipped 20 times using standard mosquito larvae dipper to sample mosquito larvae, while a D-frame dip net was used to capture the macroinvertebrate predators. Water physico-chemical parameters (dissolved oxygen, temperature, pH, conductivity, salinity and turbidity) were taken in situ at access points, while hardness and alkalinity were measured titrimetically. The influence of macroinvertebrate predator occurrence was correlated with mosquito larvae and water quality parameters using Generalized Linear Model (GLM). Predators (n=297) belonging to 3 orders of Hemiptera (54.2%), Odonata (22.9%) and Coleoptera (22.9%), and mosquito larvae (n=4001) belonging to 10 species, which included An.gambiae s.l (44.9%), Culex spp. (34.8%) and An. coustani complex (13.8%), An. maculipalpis (3.6%), An. phaorensis (1.2%), An. funestus group (0.5%), An. azaniae (0.4%), An. hamoni (0.3%), An. christyi (0.3%), An. ardensis (0.08%), An. faini (0.07%), An. sergentii (0.05%) and 0.05% of Aedes mosquito larvae which were not identified to species level, due to lack of an appropriate key, were captured from different habitats along the Mara river. It was established that invasion of habitats by the macroinvertebrate predators were partially driven by the presence of mosquito larvae (p < 0.001), and the prevailing water physico-chemical parameters (DO, temperature, and turbidity, p <0.001). Understanding abiotic and biotic factors which favour mosquitoes and macroinveterbrate co-occurrence may contribute to the control of malaria

Aggressive breast cancer in western Kenya has early onset, high proliferation, and immune cell infiltration

Background Breast cancer incidence and mortality vary significantly among different nations and racial groups. African nations have the highest breast cancer mortality rates in the world, even though the incidence rates are below those of many nations. Differences in disease progression suggest that aggressive breast tumors may harbor a unique molecular signature to promote disease progression. However, few studies have investigated the pathology and clinical markers expressed in breast tissue from regional African patient populations. Methods We collected 68 malignant and 89 non-cancerous samples from Kenyan breast tissue. To characterize the tumors from these patients, we constructed tissue microarrays (TMAs) from these tissues. Sections from these TMAs were stained and analyzed using immunohistochemistry to detect clinical breast cancer markers, including estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor 2 receptor (HER2) status, Ki67, and immune cell markers. Results Thirty-three percent of the tumors were triple negative (ER-, PR-, HER2-), 59 % were ER+, and almost all tumors analyzed were HER2-. Seven percent of the breast cancer patients were male, and 30 % were <40 years old at diagnosis. Cancer tissue had increased immune cell infiltration with recruitment of CD163+ (M2 macrophage), CD25+ (regulatory T lymphocyte), and CD4+ (T helper) cells compared to non-cancer tissue. Conclusions We identified clinical biomarkers that may assist in identifying therapy strategies for breast cancer patients in western Kenya. Estrogen receptor status in particular should lead initial treatment strategies in these breast cancer patients. Increased CD25 expression suggests a need for additional treatment strategies designed to overcome immune suppression by CD25+ cells in order to promote the antitumor activity of CD8+ cytotoxic T cells

Wash durability and optimal drying regimen of four brands of long-lasting insecticide-treated nets after repeated washing under tropical conditions

<p>Abstract</p> <p>Background</p> <p>The current study was undertaken to determine the optimal wash-drying regimen and the effects of different washing procedures on the efficacy, and durability of four brands of newly introduced long-lasting insecticide-treated nets (LLINs) under tropical conditions.</p> <p>Methods</p> <p>In the current study, the following four LLINs were tested: Olyset^®, PermaNet ^®2.0, BASF^®and TNT^®. Nets were divided into three sets; one set was washed by hand rubbing and air-dried either hanging or spread on the ground in direct sunlight or hanging or spread on the ground under the shade. A second set was washed using the WHO protocol (machine) and the third set was washed by beating the nets on rocks. The biological activities of the nets were assessed by a three-minute bioassay cone test and the residual insecticide contents were determined using high performance liquid chromatography (HPLC) procedure.</p> <p>Results</p> <p>Nets that were dried hanging under the shade retained more insecticide, 62.5% and recorded higher mortality compared to nets which were dried lying on the ground in direct sunlight 58.8%, nets dried under the shade spread on the ground 56.3%, and 57.8% for nets dried hanging in direct sunlight. It was also observed that nets washed by the standard WHO protocol, retained more insecticide and were more effective in killing mosquitoes compared to nets washed by local methods of hand rubbing and beating on rocks. There were significant differences between drying regimens (p < 0.0001) and between washing procedures (p < 0.001) respectively. However, the effect of net type was statistically insignificant. The statistical differences on individual nets were also compared, for PermaNet^®and TNT there were no significant differences observed between the four drying regimens (<it>p </it>= 0.7944 and 0.4703) respectively). For BASF and Olyset, the differences were significant (p < 0.001 and p > 0.0001).</p> <p>Conclusion</p> <p>The results of this study suggest that washing and drying regimen influence the insecticidal activity of LLINs. The standard WHOPES washing protocol underestimates the amount of insecticide washed from LLINs compared to the abrasive washing procedures that are used in the field. This suggests that there is need to educate net users to adopt a more gentle washing procedure while handling LLINs. The education should accompany net distribution campaigns.</p

The effect of repeated washing of long-lasting insecticide-treated nets (LLINs) on the feeding success and survival rates of Anopheles gambiae

<p>Abstract</p> <p>Background</p> <p>Insecticide-treated nets protect users from mosquito bites, thereby preventing transmissions of mosquito borne pathogens. Repeated washing of nets removes insecticide on the netting rendering them ineffective within a short period. Long-lasting insecticide-treated nets (LLINs) offer longer time protection against such bites because they are more wash resistant, and are preferred to conventionally treated nets. However, there is limited information on the effect of repeated washing of LLINs on the feeding success and survival of wild malaria vectors.</p> <p>Methods</p> <p>The current study evaluated the effect of repeated washing of four brands of LLINs on the feeding success and survival rates of <it>Anopheles gambiae </it>sl reared from wild strains. In this study, two- to five-day old F1s, reared from gravid mosquitoes collected from an area with a high coverage of LLINs were offered blood meals through protective barriers of the above LLINs. Mosquitoes were exposed for a period of 10 minutes each time. Nets were tested unwashed and subsequently after every 5^ththrough wash 15. After exposure mosquitoes were sorted out according to their feeding status. They were then held under normal laboratory conditions for 24 hours and mortality was scored in both fed and unfed.</p> <p>Results</p> <p>It was observed that mosquitoes did not feed through a barrier of unwashed LLINs. However, the feeding success and survival rates increased with successive number of washes and were also net brand dependant. After 15 washes, 49% of vectors succeeded to feed through a protective barrier of PermaNet 2.0 and 50% of the fed died after 24 hrs while after the same number of washes 60% of vectors succeeded to feed through Olyset brand of LLINs and all of them survived. In general, more mosquitoes survived after feeding through Olyset compared to the other four brands that were evaluated. When efficacy of individual LLINs was compared by a t-test analysis to a conventionally treated net, the results were not significantly different statistically for Olyset (<it>p = </it>0.239) and NetProtect (TNT) (<it>p = </it>0.135). However, the results were highly significant when comparison was made with PermaNet and Interceptor (BASF); <it>p </it>values 0.015 and 0.025 respectively.</p> <p>Conclusion</p> <p>The result of this study shows that repeated washing of LLINs at short time intervals using local washing methods may render them infective within a short time in preventing local vectors from feeding.</p

In-vitro activity of artemether, lumefantrine, dihydroartemisinin and piperaquine against Plasmodium falciparum clinical isolates transported in a formulated transport medium and EDTA from Chulaimbo sub-county Hospital

Introduction: Development of resistance to antimalarials has emerged as one of the greatest challenges facing malaria control today. In vitro drug sensitivity relies on the growth of plasmodium in the presence of the antimalarials. Any delay beyond 24 hours in performing the assay results in decreased parasite viability and subsequent IC50 values. Fresh isolates brought in for assay from collection sites situated far from the laboratory require a medium to keep the parasites viable in order to produce results which more accurately reflect intrinsic antimalarial drug resistance. Objective: To evaluate the use of a formulated transport medium (TM) in maintaining the viability of plasmodium during storage at 40C against a panel of antimalarials, compared to the conventional EDTA anticoagulant. Methods: This was a cross-sectional study. Blood samples positive for Plasmodium falciparum by light microscopy were transported to the laboratory using TM and the conventional EDTA anticoagulant as control.  Using 322 Plasmodium falciparum isolates from patients attending outpatient clinic at Chulaimbo sub county hospital, SYBR Green 1 in vitro assay IC50 was done against artemether, lumefantrine, dihydroartemisinin and piperaquine. Results: The mean IC50 values for drugs were not significantly different in either EDTA or TM samples. Similarly, there was no significant correlation between samples which were transported in EDTA except for the Artemisinin derivative DHA which had a significant correlation with ART (r = 0.123, p=0.03), LUM (r = 0.1382, p=0.01), and PPQ (r = 0.1281, p=0.02) and also in TM between DHA and LUM (r=0.1229, p=0.03). Conclusion: Similarity obtained in median IC50 between samples in EDTA anticoagulant and TM warrants its continued use. However, DHA significant correlation with most of the drugs used, calls for more research to expound on this finding. Keywords: Isolates, transport medium, EDTA, susceptibility, inhibitory concentration 50, resistance.

Low prevalence of Plasmodium falciparum infection among asymptomatic individuals in a highland area of Kenya

In areas of highly seasonal Plasmodium falciparum transmission, the presence of a large reservoir of persistently infected but asymptomatic individuals in the dry season leads to predictable increases in the incidence of clinical malaria in the rainy season. Highland areas, by contrast, are prone to unpredictable epidemics of malaria. To determine the importance of persistent asymptomatic infection in highland areas, we assessed asymptomatic individuals in the highland area of Kipsamoite, Kenya for the presence of P. falciparum blood-stage infection by microscopy and PCR. Five sample collections were performed during rainy and dry seasons over a 31-month period. The final collection was obtained at the start of a rainy season epidemic. Asymptomatic parasitemia was infrequent, ranging from 1.3 to 8.1% by microscopy and 5.9 to 14.5% by PCR testing. Microscopy had low sensitivity (22.2-54.8%) but excellent specificity (95.4-100%) in comparison to PCR testing. Frequency of asymptomatic parasitemia did not differ by age. Gametocyte prevalence was \u3c1% in all periods, except at the start of the epidemic, when it increased to 5.3%. In this epidemic-prone highland area, inter-epidemic periods are characterized by low frequencies of asymptomatically infected individuals. Increases in gametocyte prevalence may be an early indicator of impending outbreaks

Gamma Interferon Responses to Plasmodium falciparum Liver-Stage Antigen 1 and Thrombospondin-Related Adhesive Protein and Their Relationship to Age, Transmission Intensity, and Protection against Malaria

Gamma interferon (IFN-γ) responses to the Plasmodium falciparum antigens liver-stage antigen 1 (LSA-1) and thrombospondin-related adhesive protein (TRAP) are thought to be important in protection against malaria. Optimal methods of testing and the effects of age and transmission intensity on these responses are unknown. IFN-γ responses to LSA-1 and TRAP peptides were assessed by the enzyme-linked immunospot assay (ELISPOT) and enzyme-linked immunosorbent assay (ELISA) in children and adults from areas of stable and unstable malaria transmission in Kenya. Adults in the areas of stable and unstable transmission had similar frequencies and levels of IFN-γ responses to LSA-1 and TRAP as determined by ELISPOT and ELISA. In contrast, IFN-γ responses to the LSA-1 T3 peptide (assessed by ELISPOT) and to any LSA-1 peptide (assessed by ELISA) were less frequent in children in the area of unstable transmission than in children in the area of stable transmission. IFN-γ responses to LSA-1 were more frequently detected by ELISA than by ELISPOT in the stable-transmission area. IFN-γ responses detected by ELISA and ELISPOT did not correlate with each other. In children in the stable-transmission area, IFN-γ responses to LSA-1 peptides assessed by ELISA, but not by ELISPOT, were associated with protection against clinical malaria and anemia. IFN-γ responses to LSA-1 appear to require repeated P. falciparum exposure and/or increased age and, as measured by ELISA, are associated with protection against clinical malaria and anemia