Tumor infiltrating leukocyte density is independent of tumor grade and molecular subtype in aggressive breast cancer of Western Kenya.

BACKGROUND: Tumors commonly are infiltrated by leukocytes, or tumor infiltrating leukocytes (TILs). It remains unclear, however, if the density and type of individual TILs has a direct or simply correlative role in promoting poor prognosis in breast cancer patients. Breast cancer in Kenyan women is aggressive with presentation at a young age, with advanced grade (grade III), large tumor size (>2.0 cm), and poor prognosis. We previously observed that the tumors were predominantly estrogen receptor positive (ER+) but also included both a high percentage of triple negative tumors and also increased immune cell infiltration within the tumors. We used breast tumor tissues from each patient to make tissue microarrays that were then stained for leukocyte and myeloid markers including CD4, CD8, CD20, CD25, CD68, and CD163 using immunohistochemical techniques. The immune cell infiltration into the cancer tissue included increased numbers of macrophages (CD68+), helper T cells (CD4+), and CD25+ lymphocytes compared to benign tissue. RESULTS: This study characterized the grade, molecular subtypes, and proliferation index of these tumors and determined if TIL density was enriched across any of these factors. We analyzed 49 malignant patient tissue samples for this study. The patient population had a mean age of 51.9 years. The tumors analyzed were heterogeneous by grade: grade I (6%), grade II (47%), and grade III (39%). Most patients presented with large tumors (>2.0 cm) (69%). We classified the tumors into molecular subtypes based on clinical marker expression. Based on this analysis, the molecular subtype distribution was heterogeneous with luminal B (41%), basal/triple negative (TN) (37%), luminal A (14%) and HER2 (8%) breast cancer subtypes. While the basal/TN subtype had a much higher proliferative index (Ki-67+) than did the other molecular subtypes, we did not see a significant correlation between TIL density and either subtype or tumor grade. Therefore, TIL density is independent of molecular subtype and grade. CONCLUSION: This study identified a Kenyan patient cohort that develops large, high-grade tumors primarily of the luminal B and basal molecular subtypes. After analyzing the TILs within these tumors, we found that immune cell infiltration of these tumors correlated with increased proliferation but not grade or molecular subtype. Future research is required to determine if the aberrant recruitment of TILs to tumors contributes to cancer progression and response to cancer treatments.

Distribution and abundance of schistosomiasis and fascioliasis host snails along the Mara River in Kenya and Tanzania.

We purposively selected 39 sampling sites along the Mara River and its two perennial tributaries of Amala and Nyangores and sampled snails. In addition, water physicochemical parameters (temperature, turbidity, dissolved oxygen, conductivity, alkalinity, salinity and pH) were taken to establish their influence on the snail abundance and habitat preference. Out of the 39 sites sampled, 10 (25.6%) had snails. The snail species encountered included Biomphalaria pfeifferi Krauss - the intermediate host of Schistosoma mansoni Sambon, Bulinus africanus - the intermediate host of Schistosoma haematobium, and Lymnaea natalensis Krauss - the intermediate host of both Fasciola gigantica and F. hepatica Cobbold. Ceratophallus spp., a non-vector snail was also encountered. Most (61.0%) of the snails were encountered in streamside pools. Schistosomiasis-transmitting host snails, B. pfeifferi and B. africanus, were fewer than fascioliasis-transmitting Lymnaea species. All the four different snail species were found to be attached to different aquatic weeds, with B. pfeifferi accounting for over half (61.1%) of the snails attached to the sedge, followed by B. africanus and Lymnaea spp., accounting for 22.2 and 16.7%, respectively. Ceratophallus spp. were non-existent in sedge. The results from this preliminary study show that snails intermediate hosts of schistosomiasis and fascioliasis exists in different habitats, in few areas along the Mara River, though their densities are still low to have any noticeable impacts on disease transmission in case they are infected. The mere presence of the vector snails in these focal regions calls for their immediate control and institution of proper regulations, management, and education among the locals that can help curtail the spread of the snails and also schistosomiasis and fascioliasis within the Mara River basin.

Estimation of recent and long-term malaria transmission in a population by antibody testing to multiple Plasmodium falciparum antigens.

BACKGROUND: Tools that estimate recent and long-term malaria transmission in a population would be highly useful for malaria elimination programs. METHODS: The prevalence of antibodies to 11 Plasmodium falciparum antigens was assessed by cytometric bead assay or enzyme-linked immunosorbent assay in 1000 people in a highland area of Kenya over 14 months, during a period of interrupted malaria transmission. RESULTS: Antibodies differed by antigen in acquisition with age: rapid (>80% antibody positive by age 20 years, 5 antigens), moderate (>40% positive by age 20 years, 3 antigens), or slow (<40% positive by age 20 years, 3 antigens). Antibody seroreversion rates in the 14 months between samples decreased with age rapidly (7 antigens), slowly (3 antigens), or remained high at all ages (schizont extract). Estimated antibody half-lives in individuals >10 years of age were long (40 to >80 years) for 5 antigens, moderate (5-20 years) for 3 antigens, and short (<1 year) for 3 antigens. CONCLUSIONS: Antibodies to P. falciparum antigens in malaria-endemic areas vary by age, antigen, and time since last exposure to P. falciparum. Multiplex P. falciparum antibody testing could provide estimates of long-term and recent malaria transmission and potentially of a population's susceptibility to future clinical malaria.

Standardization and validation of a cytometric bead assay to assess antibodies to multiple Plasmodium falciparum recombinant antigens.

BACKGROUND: Multiplex cytometric bead assay (CBA) have a number of advantages over ELISA for antibody testing, but little information is available on standardization and validation of antibody CBA to multiple Plasmodium falciparum antigens. The present study was set to determine optimal parameters for multiplex testing of antibodies to P. falciparum antigens, and to compare results of multiplex CBA to ELISA. METHODS: Antibodies to ten recombinant P. falciparum antigens were measured by CBA and ELISA in samples from 30 individuals from a malaria endemic area of Kenya and compared to known positive and negative control plasma samples. Optimal antigen amounts, monoplex vs multiplex testing, plasma dilution, optimal buffer, number of beads required were assessed for CBA testing, and results from CBA vs. ELISA testing were compared. RESULTS: Optimal amounts for CBA antibody testing differed according to antigen. Results for monoplex CBA testing correlated strongly with multiplex testing for all antigens (r = 0.88-0.99, P values from <0.0001 - 0.004), and antibodies to variants of the same antigen were accurately distinguished within a multiplex reaction. Plasma dilutions of 1:100 or 1:200 were optimal for all antigens for CBA testing. Plasma diluted in a buffer containing 0.05% sodium azide, 0.5% polyvinylalcohol, and 0.8% polyvinylpyrrolidone had the lowest background activity. CBA median fluorescence intensity (MFI) values with 1,000 antigen-conjugated beads/well did not differ significantly from MFI with 5,000 beads/well. CBA and ELISA results correlated well for all antigens except apical membrane antigen-1 (AMA-1). CBA testing produced a greater range of values in samples from malaria endemic areas and less background reactivity for blank samples than ELISA. CONCLUSION: With optimization, CBA may be the preferred method of testing for antibodies to P. falciparum antigens, as CBA can test for antibodies to multiple recombinant antigens from a single plasma sample and produces a greater range of values in positive samples and lower background readings for blank samples than ELISA.

Age-related differences in naturally acquired T cell memory to Plasmodium falciparum merozoite surface protein 1.

Naturally acquired immunity to Plasmodium falciparum malaria in malaria holoendemic areas is characterized by the gradual, age-related development of protection against high-density parasitemia and clinical malaria. Animal studies, and less commonly, observations of humans with malaria, suggest that T-cell responses are important in the development and maintenance of this immunity, which is mediated primarily by antibodies that slow repeated cycles of merozoites through erythrocytes. To advance our rather limited knowledge on human T-cell immunity to blood stage malaria infection, we evaluated CD4 and CD8 T-cell effector memory subset responses to the 42 kDa C-terminal fragment of Merozoite Surface Protein 1 (MSP1(42)), a malaria vaccine candidate, by 49 healthy 0.5 to ≥18 year old residents of a holoendemic area in western Kenya. The proportion of individuals with peripheral blood mononuclear cell MSP1(42) driven IFN-γ ELISPOT responses increased from 20% (2/20) among 0.5-1 year old children to 90% (9/10) of adults ≥18 years (P = 0.01); parallel increases in the magnitude of IFN-γ responses were observed across all age groups (0.5, 1, 2, 5 and ≥18 years, P = 0.001). Less than 1% of total CD4 and CD8 T-cells from both children and adults produced IFN-γ in response to MSP1(42). However, adults had higher proportions of MSP1(42) driven IFN-γ secreting CD4 and CD8 effector memory (CD45RA(-) CD62L(-)) T-cells than children (CD4: 50.9% vs. 28.8%, P = 0.036; CD8: 52.1% vs. 18.3%, respectively P = 0.009). In contrast, MSP1(42) driven IFN-γ secreting naïve-like, transitional (CD45RA(+) CD62L(+)) CD4 and CD8 cells were the predominant T-cell subset among children with significantly fewer of these cells in adults (CD4: 34.9% vs. 5.1%, P = 0.002; CD8: 47.0% vs. 20.5%, respectively, P = 0.030). These data support the concept that meaningful age-related differences exist in the quality of T-cell immunity to malaria antigens such as MSP1.

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

BACKGROUND: 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. METHODS: The current study evaluated the effect of repeated washing of four brands of LLINs on the feeding success and survival rates of Anopheles gambiae 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 5th through 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. RESULTS: 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 (p = 0.239) and NetProtect (TNT) (p = 0.135). However, the results were highly significant when comparison was made with PermaNet and Interceptor (BASF); p values 0.015 and 0.025 respectively. CONCLUSION: 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.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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 (p = 0.7944 and 0.4703) respectively). For BASF and Olyset, the differences were significant (p < 0.001 and p > 0.0001). CONCLUSION: 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.

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 <1% 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.

Antibodies to Plasmodium falciparum antigens vary by age and antigen in children in a malaria-holoendemic area of Kenya.

BACKGROUND: Antibodies are important in protection against infection and disease caused by Plasmodium falciparum, but the frequencies of antibodies to multiple P. falciparum antigens in children are not well-characterized. METHODS: IgG and IgM antibodies to the vaccine candidate antigens circumsporozoite protein, thrombospondin-related adhesive protein, liver stage antigen-1, apical membrane antigen-1, erythrocyte-binding antigen-175 and merozoite surface protein-1 were measured by enzyme-linked immunosorbent assay in 110 children 0-50 months of age in a malaria holoendemic area of Kenya. RESULTS: A similar pattern was seen for IgG antibodies to circumsporozoite protein, thrombospondin-related adhesive protein, apical membrane antigen-1 and erythrocyte-binding antigen-175: high frequencies (70-90%) in children 0-4 months of age; a decrease in children 5-20 months of age (35-71%); and progressive increases in children 21-36 and 37-50 months of age (53-80% and 60-100%, respectively). In contrast, IgG antibodies to liver stage antigen-1 were infrequent in children 0-4 months of age (5%) and increased with age to 64%, and IgG antibody frequencies to merozoite surface protein-1 were similar across age groups (26-52%). IgG antibodies to all antigens were predominantly of the IgG1 and IgG3 subclasses. Frequencies of IgM antibodies to all antigens were low in children 0-4 months of age (0-15%) and increased with age (24-56% in the oldest children). CONCLUSION: In children in a malaria-holoendemic area, IgM antibody to all P. falciparum antigens is infrequent in the first 4 months of life but increases with age and increased exposure. The pattern of age-related IgG response frequencies to P. falciparum antigens varies significantly by antigen.

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-gamma) 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-gamma 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-gamma responses to LSA-1 and TRAP as determined by ELISPOT and ELISA. In contrast, IFN-gamma 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-gamma responses to LSA-1 were more frequently detected by ELISA than by ELISPOT in the stable-transmission area. IFN-gamma responses detected by ELISA and ELISPOT did not correlate with each other. In children in the stable-transmission area, IFN-gamma responses to LSA-1 peptides assessed by ELISA, but not by ELISPOT, were associated with protection against clinical malaria and anemia. IFN-gamma 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.

In vitro antimalarial activity of extracts of Albizia gummifera, Aspilia mossambicensis, Melia azedarach and Azadirachta indica against Plasmodium falciparum.

Since chemotherapy is presently the primary strategy of malaria control in the world, and some malaria parasites are developing resistance to the commonly used antimalarial drugs, new antimalarial compounds are required. Therefore, it is important to test antimalarial activities of medicinal plant extracts which most herbalists claim to cure malaria. We evaluated the antimalarial activities of extracts of Albizia gummiffera, Aspilia mossambicensis, Melia azedar and Azadirahchta indica against laboratory adapted isolates of Plasmodium falciparum using an in vitro radioisotopic uptake technique. Chloroquine was used as a reference antimalarial drug. Al. gummifera had the highest antimalarial activity (mean fifty percent inhibitory concentration {IC(50)S} in ug/ml of test culture =3.5 +1.6SD, n=3) followed by As. mossambicensis (mean IC(50)=29.3+11.8SD, n=4) and Me. Azedarach (mean IC(50) =299.7+202.0SD, n=4). And lastly Az. Indica (mean IC(50)=349.9+213.1 SD, n=4). The antimalarial activities of the reference drug, chloroquine, was far much higher (mean IC(50)=0.065+0.057SD, n=)4). These findings show that Al. gummifera and As. mossambicensis plant extracts have potent antimalarial compounds. Phytochemical analyses should be done on these two plants to isolate the compound(s) containing he active principles(s).