PHD-Department of Pathology

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The swain-langley and McCoy blood group polymorphisms of complement receptor 1: role in severe plasmodium falciparum malaria
(Kenyatta University, 2009-11) Guyah, Bernard Ochieng'
Expression of fcyrilll, cr3 intracellular tumor necrosis factor-alpha and nitric oxide production by monocytes from children with plasmodium falciparum malaria
(2011-07-19) Ogonda, Adhiambo L.
Malaria is a major cause of childhood morbidity and mortality in tiub-Saharan Africa. Most of the mortality is the result of severe Plasmodium falciparum (P. falciparum) disease complications such as severe malarial anemia (SMA) and cerebral malaria (CM). A clear understanding of the factors that play a role in the pathogenesis of severe P. falciparum malaria is essential for the development of effective prophylactic and therapeutic measures. Malaria infection leads to formation of immune complexes (ICs) that can interact with monocyte/macrophages by binding to their surface Fc gamma and complement receptors. The interaction of ICs with Fe gamma receptor III (FcyRIII, CD16) on monocytes/macrophages results in stimulation leading to production of nitric oxide (NO) and the cytokine tumor necrosis factor-alpha (TNF-(X) which ire important factors implicated in the development of severe malaria. FcyRIIIA can also mediate phagocytosis of antibody-coated infected and uninfected red cells which could contribute to the development of severe anaemia. However, phagocytosis of opsonized ICs via complement receptor 3 (CR3, Mac-1, CD I Ib/CD18) suppresses pro-inflammatory monocyte functions such as NO production. Therefore, the expression levels of these receptors on monocytes/macrophages may influence degree of stimulation and determine individuals' susceptibility to severe malaria. This study examined the expression of FcyRIII and CR3 on monocytes of children with severe malarial anaemia, cerebral malaria, and their age and gender-matched uncomplicated malaria controls by flow cytometr y. Whole blood was obtained from the patients during the acute illness and after recovery from illness and stained with directly conjugated antibodies against CD14, FcyRIII/CD16 and CR3/CD1 lb followed by red cell lysis. In addition, monocytes were stimulated with BSA-anti-BSA immune complexes to determine the effect on the intracellular expression of TNF-a and NO by monocytes. The expression of FcyRIII on total CD16+ mono~cyte population and on the CD14+CD16+ monocyte subpopulation was highest in SMA vases and correlated negatively with the haematocrit levels. The expression of CR3 w CD14+CD16+ and CD14++CD16+ monocytes sub-populations was lowest in SMA ases and correlated positively with the haematocrit levels. The ability to produce N in response to IC correlated positively with age and was higher in the SMA than in ( VI group (P= 0.013) but there were no differences between these severe malaria groups 'A i th their age matched controls. Intracellular TNF-a expression by CD14+CD16+ id CD14++CD16+ monocytes in response to IC stimulation correlated positively with their FcyRIII expression levels. The concentration of total monocytes and their subpopulations was significantly higher in SMA cases than in all the other clinical groups and correlated inversely with haematocrit levels. These findings suggest that CD14+CD16+ and CD14++CD16± monocytes may be more mature and responsive to activation in SMA cases than in CM cases. Over expression of FcyRIII on monocytes in response to P. falciparum infection may be an important contributing factor to the development of severe malarial anaemia.
Isolation of recurrent mycobacteium isolates from sputum smear negative relapse at the central reference tuberculosis laboratory in Nairobi, Kenya
(2011-08-15) Wahogo, Josephine N; Orago, Alloys S.S.; Kabiru, Ephantus W.
Pregnant women have increased susceptibility to malaria infection. In these women, malaria parasites are frequently found sequestered in the placental intervillous spaces, a condition referred to as placental malaria (PM). Placental malaria threatens the health of the mother and the child's life by causing intrauterine growth retardation, abortions, still births and reduction in gestational age. An estimated 24 million pregnant women in SubSaharan Africa are at risk. Mechanisms responsible for increased susceptibility in pregnant women are not fully understood. Baboons are susceptible to Plasmodium knowlesi and have similar host pathogen interactions and reproductive physiology similar to humans, making them attractive for the development as a model for studying mechanisms underlying development of placental malaria. This study exploited the susceptibility of baboons to Plasmodium knowlesi infection to develop a non-human primate (baboon) model for studying PM. The main objective of the study was to demonstrate PM and characterize immunological mechanisms underlying the pathogenesis of PM in baboons infected with Plasmodium knowlesi. The pregnancies of three time mated adult female baboons and their gestational levels (one in its second trimester and two in their third trimester) were confirmed by ultrasonography. On the 150th day of gestation, the pregnant baboons were infected with Plasmodium knowlesi H strain parasites together with four non pregnant controls. Peripheral parasitaemia development was monitored on a daily basis from two days post inoculation. Collection of sera, plasma, mononuclear cells and haematological samples was done on a weekly basis. Peripheral blood mononuclear cells (PBMC) were stimulated in culture with concanavalin A and P. knowlesi antigens and their proliferation quantified. Sera cytokine and immunoglobulin concentrations were measured by ELISA using respective enzyme conjugated antibodies. Two pregnant baboons aborted (one on day 6 and the other on day 7 post infection) and cesarean section was only done on one baboon. Smears prepared from placental blood demonstrated the presence Plasmodium knowlesi parasites in all the sampled placentas. On average, the pregnant baboons had more than 29 fold higher placental parasitaemia than simultaneous peripheral parasitaemia. This shows that Plasmodium knowlesi preferentially sequesters in the baboon placenta just like Plasmodium falciparum does in humans. Two baboons that had high placental parasitaemia experienced abortion, which is a sequele of human placental malaria. Results indicate that PM in this model is associated with significant (P < 0.05) suppression of immunoglobulin G, Interferon gamma, and interleukin 6 responses. Tumour necrosis factor alpha responses were significantly upregulated (P < 0.05) while immunoglobulin M, interleukin 10, interleukin 12, interleukin 4 and PBMC proliferation responses did not differ from controls (P > 0.05). These data are consistent with some findings from human studies, showing the feasibility of this model for studying mechanisms underlying placental malaria. The study has contributed valuable data to be used in further studies and the development of preventative, control and therapeutic measures against PM in women
Consistency of stools as an indicator of type of intestinal parasites harboured by patients with abdominal pain at Kenyatta National hospital, Nairobi, Kenya
(2011-12-02) Githigaro, Peter Mathenge
Abdominal pain is a common problem reported to clinicians for various ailments. Hospital records in Kenya show that high percentage of patients suffers from abdominal pain. Among the causes of abdominal pain are intestinal parasites. Intestinal parasites include protozoa and helminthes living in the intestine of animals and human. They are among the most common infections in developing countries. About 3.5 billion people worldwide are infected with intestinal parasites without considering those with abdominal pain. There are many people living with intestinal parasites without knowing. The impact of these parasites on public health has been under estimated, although they cause considerable morbidity and mortality. The main objective of this study was to determine whether consistency of stool could be used as an indicator of type of intestinal parasites causing abdominal pain in a patient. Patients being investigated for intestinal parasitic infections in the clinical laboratory were interviewed and a sample size of 400 patients suffering from abdominal pain selected. Stools of patients were macroscopically examined to determine their consistency; both direct saline and formal-ether concentration methods were used to identify parasitic agents. Stool specimens with eggs of Taenia species were stained and eggs differentiated to species using modified Ziehl-Neelsen stain technique. Harada-Mori culture technique was done on positive stools with hookworm looking like eggs in order to differentiate the worm species. Data was analyzed, and association among stool consistency, age groups, sex and intestinal parasites were tested using chi-square. Of the 400 patients 164(42.0%) had acute abdominal pain, 181(45.3°/x) had chronic abdominal pain and 55(13.8%) had recurrent abdominal pain. The consistency of stools from those patients were 218(54.5%) formed, 95(23.8%) soft, 78(19.5%) loose and 9(2.3%) watery. Stools from 79(19.75%) patients had inclusions; those with pus cells were 44(11.0%), mucus 33(8.25%), blood 23(:5.75%) and yeast cells 15(3.75%). The results show 27.5% of patients complaining of abdominal pain had intestinal parasitic infections. The most prevalent helminth was hookworm (2.5%) while the most prevalent protozoan was Blastocystis hominis (10.75°/). The study show stools from patients complaining of acute abdominal pain had higher amount of water than stools from patients with ether chronic or recurrent abdominal pain (x2=47.3, df=6, P=0.000). Patient's complains of different duration of abdominal pain was not related to presence of intestinal parasitic agents in stools (x2=72.55, df 64, P=0.207). There was no association between intestinal parasitic infections and consistency of stools (x2=0.000, df 3, P=1.000). Patients of 36-40 years age group had the highest number 50% of parasitic infection eases than any other age group (x2=38.52, df=10, P<0.001). The findings from this study provide a general awareness among KNH practicing physicians the need of laboratory diagnosis of stools, in confirming clinical findings when treating patients with abdominal pain.
Anti-leishmanial activity of acacia mellifera (leguminosae: momosoideae) against leishmania Major
(2011-12-02) Mburu, Faith Wambui
Leishmaniasis is a major group of parasitic diseases in the tropical regions. Their public health importance requires integrated measures in order to ensure effective control of the disease. Chemotherapy is the mainstay of control with pentavalent antimomal agents still widely used as the drugs of choice. However. these drugs are associated with many undesired effects, which include renal and cardiac toxicity. nlyalgia, anthralgia. pancreatitis, gastrointestinal problems. cardiac arrhythmia and sudden death. I he antimonials are also very expensive, and require a long period of administration. Subsequently. the search for compounds with possible antileishmanial activity is growing. A lot of locus is currently being given to natural products. It has long been known that plants are a rich source of compounds with potential medicinal uses. ,Acacia mellifera subsp. mellifera is a recognized medicinal plant used for conditions such as malaria pneumonia, sterility, stomach problems and primary stages of syphilis. However_ limited information is Available on its isolated metabolites. The main objective of this study was to determine whether A. mellifera has any antileisllnlanial activities. To achieve this. the stem bark and leaves of .A. mellifera were extracted using methanol and dichloromethane then extracts evaluated against Leishmania major promastigotes and alnastigotes in limo. Further fractionation on dichloromethane extracts yielded nine fractions designated as fractions I-IX. The effects of extracts as well as fractions were evaluated both in cell free media and using infected macrophages. To evaluate the immunostimulatorv activities of the extracts, production of nitric oxide in supernatants from macrophages stimulated with extracts was detected by Griess test. All tests were done in triplicate. Statistical analyses of the results obtained were done by analysis of variance. chi-square and Kluskal-Wallis test. Results revealed that extracts containing a combination of methanol and dichloromethane had the highest activity against L. major promastigotes in cell-free culture. 100%) mortality was observed on promastigotes cultured with 5mg/ml of the methanol and dichloromethane crude extracts. The methanol and dichloromethane stem bark extracts, however. did not inhibit the transformation of L. major alnastigotes into promastigotes (P>0.05). The stem bark methanol extract had the lowest infection rate of 41.35%. Infection rates of' the extracts compared to a positive control had a difference (x2 =13.89; df=9: P>0.05). The fractions of dichloromethane had a statistically significant difference in their infection rates (F=6.827: df=4.15: P<0.01). Fraction VII had the lowest infection rates of 30% at 250mg/ml and also the lowest IC50 of' 54.19pg/ml. Close in its activity were fractions Vlll and IX. Results for immunostimulatory activity among the fractions of dichloromethane indicated that there was no significant production of nitric oxide (P>0.05). These results suggest another possible mechanism of action of the fractions other than through stimulation of nitric oxide production. A comparison of the immunostimulatory effects between the methanol and dichloromethane indicated production of nitric oxide by the methanol extract. There was a difference (H4,4,4,4= 14.04: P<0.05). The results from this study demonstrate that Acacia mellifera indeed has activity against Leishmania major parasites. This is a major contribution toward attainment of the Millenium Development Goal that seeks to combat disease.
Transmission dynamics of Human Rift Valley Fever virus in Ijara District, Garrissa county, Kenya
(2012-04-13) Muiruri, Samuel Kinyanjui
Rift Valley Fever (RVF) is an emerging viral disease which causes among other problems severe hemorrhagic syndrome, retinitis and meningo-encephalitis in humans. The disease was previously thought to be a disease of animals but it is increasingly developing to be a human health problem. Two human RVF epidemics have previously occurred in Kenya (1997-1997; 2006-2007) each resulting to human deaths. Most RVF outbreaks occur in rural locations where keeping of livestock is the main livelihood of the affected communities. Flooding due to excessive rainfall after a long dry spell allows the breeding of vector mosquitoes. Human beings get infected either through the bite of infected mosquitoes or exposure to infected animal material. The dominant mode of transmission of RVF from animal to human during combined epizootics and epidemics has not been clearly understood. The main objective of this study is to assess the transmission dynamics of R VF in an area where epidemics occur. The survey was done in Gumarey, a rural village and Sogan-Godud, a sub-urban village in Ijara District, Kenya where 248 study participants were randomly selected. Venous blood was then collected fromthesubjects for the detection of Rift Valley Fever Virus (R VFV)antibodies by IgG Enzyme Linked Immunosorbent Assay (ELISA) testing technique. Rift Valley Fever Virus (RVFV) IgG seropositivity was used as the primary measure for RVFV exposure. Animal and non- animal exposure factors were then determined against RVFV IgG seropositivity. Subjects were clinically examined for ophthalmological disorders by an Ophthalmologist. A structured questionnaire was used to collect relevant data while observations were made on the homesteads. Mosquito sampling was done by use of Centers of Disease Control (CDC) Light Traps. A total of 12252 mosquitoes were collected. The morphological identification was confirmed by Polymerase Chain Reaction (PCR). The mosquito RVF viral infection rates were also confirmed by PCR. The Geographical Positioning System «iPS) of the households under study was recorded by Etrex GPS machine. Overall anti-RVFV seropositivity by IgG was 13%. Increased IgG seropositivity was found among older male persons and evidence of interepidernic RVFV transmission was detected. In location analysis, participants who resided from Gumarey were 4 times. more at risk of RVFV than those residing from Sogan Gudud (P=O.OOl;Odds Ratio: 3.8). Gumarey village reported more animal exposures than Sogan Gudud. The results also showed significant association between eye disease and seropositivity (P=O.003) and the most common causes of eye disease in seropositive persons were visual impairments and retinal lesions. Disposal of animal fetus, birthing, skinning, butchering and milking were highly associated with seropositivity (p=O.OOOl). In total22% of the mosquito pools tested by peR were positive for R VFV. A geospatial map showing seropositive households by clusters was constructed for both villages. The study highlights the variability in exposure and anti-R VFV seropositivity between Villages and also emphasizes the impact of age, gender, location and animal husbandry in RVF transmission. The results show the potential of future outbreaks in Ijara District. Public health education could be used to prevent transmission ofRVF and avert possible epidemics.
Plant-feedingg behaviour and its effects on the fitness and competence of the malaria vector anopheles Gambiae (diptera: Culicidae)
(2012-06-22) Manda, Hortance
Malaria remains a serious threat in sub-Saharan Africa Available control tools are now largely ineffective. Because of the role of vectors in malaria transmission, integrated vector management strategies that are environmentally safe, economically feasible and acceptable to resource-limited communities are one way to achieve effective malaria control. However, adequate knowledge of the biology, behaviour and ecology of the malaria vectors are imperative. Sugar-feeding from plants is a basic requirement for adult mosquitoes, and so far have received little attention. The present study, conducted in Suba district (a malaria endemic area of western Kenya), aimed to investigate the plant-feeding behaviour and its effects on the fitness and competence of Anopheles gambiae. Females of two species of Anopheles gambiae s. l. namely Anopheles gambiae s.s., and Anopheles arabiensis were sampled during both dry and wet seasons. Approximately 12% were fructose-positive at the time of collection, indicating recent plant-feeding. Proportions of plant-fed mosquitoes were similar in all the 2 Anopheles species (P=0.90), at all their gonotrophic status (unfed, blood-fed, half-gravid, and gravid) (P=0.69), at all ages (parous and nulliparous) (P=0.98), and during both seasons (P=0.06). However, more mosquitoes had fed on plants when collected far from larval habitats (P=0.001). When offered 13 dominant plant species [Cassia hirsuta L., Senna bicopsularis L., Senna didymobotrya F. (Leguminosae), Daturo stramonium L. (Solanaceae), Flaveria trinervia M., Parthenium hysterophorus L., I'siada punctulata V., Tithonia diversifolia H. (Asteraceae), Hamelia patens J. (Rubiaceae), Ipomea hildebrandtie V. (Convolvulaceae), Lantana camara L. (Verbenaceae), Ricinus communis L. (Euphorbiaceae), Tecoma stans L. (Bignoniaceae)) growing around human dwellings and mosquito larval habitats in the study area, behavioural responses of mosquitoes to those candidate plants were recorded in the greenhouse, each plant being presented to mosquitoes singly, and concurrently with other plants species in the no-choice and choice bioassays respectively. Gas chromatography analysis was carried out to determine and compare plants and mosquito sugar profiles. Five plants species were found to be most preferred by Anopheles gambiae, namely; Ricinus communis L., Senna didymobotrya F., Parthenium hysterophorus L., Tecoma stans L., and Hamelia patens J (P<0.001). Flowers were most fed on by mosquitoes, but leaves were most preferred in two plants species (Lantana camara and Parthenium hysterophorus); leaves and stems in Ricinus communis. With the exception of one plant species (Parthenium hysterophorus), survival (P<0.001) and fecundity (P=0.01) were high in the mosquitoes which fed on most of the preferred plants, compared to those which fed on the less preferred plant. Sugar composition and concentration in the preferred plants were positively correlated to the survival (r=1, P<0.001) and fecundity (r-0.87, P=0.04) of mosquitoes. Four plants species (Lantana camara, Senna didymobotrya, Ricinus communis and Parthenium hysterophorus) significantly reduced the prevalence of infection in mosquitoes (P<0.01). There was also a drastic decrease on the parasite load (oocyst intensity) in the midguts of the infected mosquitoes which fed on 3 plants species (Lantana camara, Senna didymobotrya and Pathernium hysterophorus) (P<0.01). The inhibitory effects of those plants were more pronounced when mosquitoes plant-fed throughout (before and after) an infectious blood meal, followed by plant-feeding post-infection. The greatest impact of deleterious plants was found in the transitions macrogamete-ookinete (only in pre-infection plant-feeding) and ookinete-oocyst (only in post-infection plant-feeding). These studies imply that certain plant species preferred by Anopheles gambiae in the endemic area significantly decrease their fitness and competence to develop Plasmodium falciparum, and therefore can reduce malaria transmission.
Natural monocytic acquisition of haemozoin and rantes polymorphisms: association with malarial disease outcomes and functional changes in children from Western Kenya
(2013-01-30) Were, Tom; Alloys Sigar S. Orago; Otieno, Micheal Frederick; Douglas Jay Perkins
Plasmodium falciparum is an important cause of morbidity and mortality in children residing in holoendemic transmission areas largely from severe malarial anaemia (SMA). Although overproduction of inflammatory-derived cytokines are implicated in the immunopathogenesis of severe malaria, the chemokine regulated on activation, normal T-cell expressed and secreted (RANTES, CCL5) is largely unexplored in the context of malaria. Additionally, pro-inflammatory cytokines, such as tumour necrosis factor (TNF)-α, interleukin (IL)-1 and interferon (IFN)- promote increased RANTES production, while anti-inflammatory cytokines (e.g., IL-4, IL-10 and IL-13) down-regulate RANTES biosynthesis. However, the effect of these cytokines on RANTES production in children with malaria anaemia (MA) is poorly understood. Although genetic variation in RANTES is associated with inflammatory, autoimmune and infectious diseases, the role of single nucleotide polymorphisms (SNPs) in conditioning disease outcomes and RANTES production in malaria remains unexplored. These studies investigated the relationship between circulating RANTES with MA severity, thrombocytopaenia, suppression of erythropoiesis, and naturallyacquired haemozoin (pfHz) in monocytes. The functional influence of promoter (403G/A, -4120A/T, and -415]C/T) and intronic (+307A/G) SNPs on malaria outcomes was also examined. These hospital-based cross-sectional studies were performed in infants and young children (age <36mos.) enrolled at Siaya District Hospital, western Kenya. RANTES levels in circulation and from peripheral blood mononuclear cell (PBMC) cultures were measured by a 25-plex cytokine assay and enzyme-linked immunosorbent assay, respectively. Genotyping of -4120A/T, - 4151C/T and +307A/G polymorphisms was performed using Taqman® 5'-allelic discrimination assay, while -403G/A was genotyped by polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) method. Results presented here show that suppression of circulating RANTES is associated with increasing severity of MA (p=0.002), decreased erythropoiesis (p=0.049), and malaria-induced thrombocytopaenia (p=0.036). Additional results demonstrate that suppression of circulating RANTES is associated with increasing levels of pigment-containing monocytes (PCM) (p=0.035). In vitro experiments indicate that monocytic acquisition of pfHz is associated with suppression of RANTES from PBMC under both baseline (p=0.001) and stimulated conditions (p=0.072). Although high levels of pfHz in monocytes caused reduced production of IFN- (p=0.003) and IL-10 (p=0.010), multivariate modelling revealed that only PCM (p=0.048, =-0.171) and IL-10 (p<0.0001, =-0.476) were independently associated with RANTES. Subsequent experiments in cultured PBMC from children with naturally-acquired Hz revealed that blockade of endogenous IL-10 caused significant increases in RANTES production (p=0.028). Haplotypic constructs of RANTES (+307/-403/-4120/-4151) revealed higher frequencies of SMA (Hb<5.0g/dL) in AGTC carriers (75.0% vs. 21.5%, p=0.010) and lower prevalence of thrombocytopaenia in AATC carriers (15.4% vs. 84.6%, p=0.036). Multivariate logistic regression analyses illustrated increased susceptibility to SMA (Hb<5.0g/dL) in AGTC carriers (OR, 13.4; 95% CI, 1.3-133.6; p=0.027) and protection from thrombocytopaenia in AATC carriers (OR, 0.2; 95% CI, 0.1-1.0; p=0.05). Moreover, circulating RANTES levels were lower in AGTC (p=0.049), and higher in AATC carriers (p=0.043). Taken together, these results demonstrate that thrombocytopaenia and natural monocytic acquisition of pfHz are a source of reduced RANTES that may contribute to suppression of erythropoiesis in children with MA. Additional results presented here demonstrate that genetic variation in RANTES is important for mediating SMA and thrombocytopaenia during P. falciparum infection. These findings have the potential to contribute to better understanding of SMA pathogenesis, and lead to better disease management modalities and/or vaccine development.
Efficacy of Crude Extracts from Allium Sativum, Callistemon Citrinus and Moringa Stenopetala of Kenya against Leishmania Major
(2014-02-22) Kinuthia, Geoffrey Kariuki; Kabiru, Ephantus W.; Gikonyo, Nicholas K.; Anjili, C. O.
Cutaneous leishmaniasis (CL) is endemic in more than 80 countries worldwide and it causes skin ulcers and disfigurement. Leishmania major causes CL in Kenya and its drugs are expensive, toxic and require prolonged use. Multidrug combination therapy prevents drug resistance and reduces toxicity. Herbal extracts can be safe and cheaper. This study investigated in vitro and in vivo efficacy of single and blends of crude aqueous and methanolic extracts from Moringa stenopetala, Callistemon citrinus, and Allium sativum against L. major. Controls were contemporary Leishmania drugs pentostam and liposomal amphotericin B, and phosphate buffered saline. Dry ground test materials were soaked in H2O at 70oC for 1½ hours, filtered, and freeze dried. Similarly, ground test materials were soaked in 500 ml of analytical grade methanol for 72 hours at room temperature, filtered and concentrated using rotary evaporator. T-test and ANOVA were used for data analysis. P-value of < 0.05 was considered significant. The minimum inhibitory concentrations (MICs) of aqueous extracts of M. stenopetala (A), C. citrinus (B), and A. sativum (C) ranged from 3 to 5mg/ml while IC50 from 297.79 to 575.75μg/ml against L. major promastigotes as compared to MICs of 12.50 and 6.25μg/ml and IC50 of 0.26 and 0.82μg/ml for pentostam and liposomal amphotericin B respectively. MICs of methanolic extracts of M. stenopetala (H), C. citrinus (G), and A. sativum (F) ranged from 1 to 5mg/ml with IC50 of 572.69 to 1752.92μg/ml against L. major promastigotes. The extracts’s cytotoxicity against vero cells ranged from 467.11 to 2105.93μg/ml as compared to 60.95μg/ml and 108.58μg/ml for pentostam and liposomal amphotericin B respectively. Methanolic extracts of C. citrinus and aqueous A. sativum extracts stimulated production of 20μM nitric oxide in BALB/c mice peritoneal macrophages signifying their immuno-modulatory role. Blends of M. stenopetala & C. citrinus (AB), M. stenopetala & A. sativum (AC) and C. citrinus & A. sativum (BC) at concentrations based on MICs of individual extracts, were active at ratios 1:1, 1:9 and 1:1 with promastigotes’ viabilities of 33.82%, 17.41% and 60.74 % respectively. The ratios and promastigotes viabilities for methanolic blends were, FG (1:1; 31.32%), FH (1:9; 34.59%) and GH (9:1; 7.44%). The IC50 for any blends of two extracts ranged from 174μg/ml to 1314μg/ml against L. major promastigotes. There was strong synergistic (1:9) and additive (1:1 and 2:8) interactions for the blend AC. Blend BC interacted additively at ratio 1:1. Blend AC at 125μg/ml had infection rate (IR) of 71% and multiplication index (MI) of 48.20% for L. major amastigotes in vitro, and compared well to pentostam at 12.50μg/ml with IR of 67% and MI of 47.51%. Methanolic blends of three different extracts were more efficacious with MIs of 33.48 to 38.24%. Oral aqueous and methanolic A. sativum extracts (A and F) reduced the foot pad lesion sizes significantly (P < 0.05) in infected BALB/c mice. Oral blend BC reduced the footpad lesion size significantly (P < 0.05) like Leishmania control drugs. Oral blends BC and AC reduced spleen amastigotes in mice by 48.33% and 60.94% corresponding to total LDUs of 6.35±0.66 and 4.80±0.95 respectively. Oral/ip blend HGF (2:2:1) had amastigotes inhibition rate of 63.95% compared to 66.40% and 60.62% for pentostam and liposomal amphotericin B respectively. In conclusion, aqueous and methanolic crude extracts of C. citrinus, A. sativum and M. stenopetala were less toxic but active against L. major in vitro and in vivo and their blends that had additive or synergistic interaction lowered L. major survival. This study recommends that Kenyatta University in collaboration with relevant stakeholders to consider developing natural products based on these results for the management of CL in poverty stricken leishmaniases endemic areas of Kenya.
Antimicrobial Resistant Escherichia Coli Genes in Children Aged Below Five Years Presenting with Diarrhoea at Thika Level 5 Hospital, Kiambu County, Kenya
(Kenyatta University, 2015-10) Kang'ethe, Kamwati Stanley
Diarrhoea is one of the leading causes of illness and death among children in developing countries, where an estimated 1.3 billion episodes and 4 to 10 million deaths occur each year in children less than 5 years of age. The study envisioned to determine the genetic basis of extended spectrum β-lactam (ESBL) resistance in E. coli isolated from children aged below 5 years attending Thika Level 5 Hospital Kiambu County in Kenya. Faecal samples were obtained from 384 children presenting with diarrhoea. A questionnaire was used to collect data concerning the age of the child, gender and additional information recorded included on history of hospitalization and antibiotic use. Samples were sub-cultured on MacConkey agar and suspect E. coli isolates identified. Antimicrobial susceptibility profiles were determined for the most common antimicrobials against Gram-negative bacteria. The types and prevalence of Extended-Spectrum βeta-Lactamases were also determined. Polymerase Chain Reaction methods were used for detection of the Extended-Spectrum βeta-Lactamases genes and carriage of integrons. Conjugation experiments were performed to determine the potential of horizontal transfer of resistance genes. The plasmid content for defining diversity of plasmid replicon types was also determined among donors and transconjugants. Approximately 25% of all isolates were resistant to ampicillin, tetracycline, chloramphenicol, streptomycin, amikacin sulphamethoxazole-trimethoprim and amoxicillinclavulanic acid (p=0.0051; OR=2.27; 95% CI: 2.01-2.33). Isolates (50%) had a Minimum Inhibitory Concentration mode50 value within the resistance range except for ceftazidime, Gentamicin, chloramphenicol, Nalidixic acid, Ciproflocin and Kanamycin. Factors identified to predispose children to carry highly resistant isolates included recent history of hospitalization and antimicrobial use and having acute diarrhoea (p=0.0026; OR=4.21; 95% CI: 3.76-4.98 ) There was no significant difference in antimicrobial resistance patterns for isolates from rural and urban children (p=0.049). Carriage of a combination of blaCTX-M+ blaTEM or a combination of blaCTX-M+blaOXA genes was highly associated with resistance to multiple β-lactams (p=0.00015; OR=2.31; 95% CI: 1.99-2.65). Isolates with multiple β-lactamases were also likely to carry integrons encoding resistance to other antimicrobials. Plasmids that mediated resistance to most antimicrobials belonged to incL/M, HI2 and F-type. In conclusion, most of isolates recovered from these children were resistant to first line treatment for diarrhoea and therefore a need to revise the existing empiric treatment of E. coli infections. Resistance was transferrable via conjugation and integrons were significantly implicated in resistance dispersion hence increase in the prevalence of multidrug resistant isolates (p=0.0017; OR: 4.03; 95% CI: 3.81- 4.04). There is a need to encourage parents and doctors to follow the treatment guidelines for diarrhoea in which case, the oral re-hydration therapy zinc and vitamin C rather than antibiotics should be administered in cases of watery bacillary diarrhoea, implement proper antimicrobial use polices and launch national surveillance programs to monitor emergence and spread of MDR isolates.
Haematological reference intervals for adolescents and adults in nakuru county, Kenya
(Kenyatta University, 2018-05) Gachie, Rose Nyambura
The haematologic reference intervals are important in the assessment of health and diseases conditions in a certain population and they vary with to age, sex, altitude and genetic. The current study intended to establish the haematological reference intervals in Nakuru County, Kenya. A cross sectional design was used where samples of blood were collected from consenting participants. The number of participants recruited for the study was 627 and they aged 13 to 18 years for the adolescents and 19 to 55 years for the adults. The erythrocyte median and reference indices (RBC count, P <0.0001; Hb, P <0.0001; HCT, P <0.0001; MCH, P =0.034; and MCHC, P <0.0001) were significantly different across the study groups. Post-hoc analyses showed that the median and reference HB, RBC, MCH, HCT, and MCHC counts were lower in female adults compared to male adults (P <0.05). Furthermore, median and reference values for MCHC were lower in the female adults compared to male adults, female adolescents and male adolescents (P <0.0001, respectively). The median and reference for Hb, MCH, RBC, Hct, and MCHC values in the male adults were however, higher in comparison to female adolescents (P <0.0001, respectively) while Hct was also lower in comparison to male adolescents (P <0.01). Finally, the median and reference Hb, Hct, RBC and MCH values were lower in the female adolescents compared to the male adolescents P <0.0001 for all). The median and the reference levels for the absolute monocyte and eosinophil counts showed statistical significance (monocyte, P <0.0001; eosinophil, P <0.001). Consequent analysis indicated differences with females adolescents having higher values than male adolescents p, <0.0001 for monocytes counts. The females indicated higher median and reference differential count levels for [lymphocytes (P<0.012), monocytes (P<0.031) and basophils (P<0.0001) than male and the values showed statistical significant (p <0.05). Post hoc indicated statistical differences within study groups with females having higher median and higher reference values for a lymphocytes and monocytes than the males adults (P<0.05) and male adolescents (P <0.0001). Age differences were also noted for lymphocytes, monocytes and basophils with female adults having lower values than the adults (P<0.0001). Females had higher median levels for absolute platelet counts than males and there was statistical difference (P <0.0001). Adult males also showed higher reference values than the male adolescents (P <0.0001). MPV also illustrated higher medium levels in females than males for adolescent (P <0.05). Further analysis indicated statistical significance with females having higher values (P <0.0001). Age differences were noted with male adults versus male adolescents for MPV (P <0.05). The results indicated low haematological reference values as compared to the currently utilized values by the clinicians in Kenya and also in Nakuru County. These data will therefore provide haematological reference values for this specific region which can also be interpolated in other regions for correct patients’ management and for clinical care.
Pathological Characteristics of Prostate Carcinoma in Archival Tissues at the Africa Inland Church Kijabe Hospital, Kiambu County – Kenya
(Kenyatta University, 2019-06) Nzioka, Ancent Kituku
Prostate carcinoma (PCa) is the leading cancer among middle and elderly men worldwide. It accounts for 15% of all cancer cases and 6.6% of all cancer associated-deaths in men. PCa is a leading cause of cancer burden among men in Africa with Kenya reporting an incidence of 16.6/100,000. The exceedingly high burden of PCa is largely due to lack of population-based screening and delayed diagnosis in the background of genetic and environmental predisposing factors. Histopathological features of PCa offer prognostic indicators while immunohistochemical (IHC) staining provide insights into the molecular characteristics of PCa which have both prognostic and predictive value in the management of PCa. The androgen receptor (AR), Human epidermal growth receptor (HER2/neu), and protein p53 genes are among the most important genes regulating PCa pathogenesis. IHC staining patterns of these gene products have been associated with different therapeutic and prognostic outcomes. This cross-sectional study determined the histopathological and AR, HER2 and p53 IHC expression on archival simple prostatectomy specimens (n=210) at AIC Kijabe Hospital, a tertiary faith-based health facility in Kiambu County, Kenya. Patient demographic and gross characteristics of the prostatectomy specimens were obtained from the patient files. The tissue blocks (n=210) were retrieved, sectioned and stained using standard H&E and immunohistochemical staining protocols. The slides were then examined using light microscopy and histopathological and immunohistochemical features recorded. Data analyses showed that the median age of the subjects was 74.0 years (44.0 – 99.0). Ethnic groups were represented as follows; Bantu (68.0%), Nilotes (31.0%) and Cushites (1%). Histopathologically, 97.6% of the PCa were of acinar adenocarcinoma type with the rest being of ductal adenocarcinoma type. Perineural invasion (PNI) was present in 57.6% of the PCa while 25.2% had lymphovascular invasion (LVI). Low grade PCa (Gleason score <8) constituted 56.6 % of the cases while high grade PCa (Gleason score ≥8) constituted 43.4% of the cases. The frequencies of the different ISUP grade groups were as follows; ISUP grade 1 (23.3%), grade 2 (16.2%), grade 3 (17.1%) grade 4 (10.5%) and grade 5 (32.9%). This study showed that 34.8% of the PCa were AR negative, 4.5% had HER2 over-expression while 19.7% of the cases showed p53 protein over-expression. Binary logistic regression analyses revealed that Gleason score ≥8 was associated with higher odds for PNI (OR, 40.407; 95% CI, 11.036-147.949; P<0.0001) and gland involvement of ≥50% (OR, 30.447; 95% CI, 7.220-128.397; P<0.0001), but lower odds for AR expression (OR, 0.109; 95% CI, 0.019-0.621; P=0.013). In addition, PCa pathological grade III was associated with PNI (OR, 37.710; 95% CI, 8.762-162.288; P<0.0001), LVI (OR, 33.531; 95% CI, 13.757-81.729; P<0.0001), and gland involvement of ≥50% (OR, 2.291; 95% CI, 1.040-50.500; P=0.040). Likewise, p53 expression was associated with a lack of PNI (OR, 0.157; 95% CI, 0.032-0.778; P=0.023). Altogether, this is the first PCa study outlining the pathological characteristics of PCa in Kenya. This is the first African study that has used ISUP grade groups for PCa. These findings will influence the diagnosis, treatment and prognosis of PCa and might become the standard of care in the country. Further studies to compare patient characteristics, biochemical test results and pathological findings of PCa are recommended.
In Vitro Antibacterial, In Vivo Immunomodulatory and Safety Properties of Ethyl Acetate Leaf Extract of Ocimum Basilicum
(Kenyatta University, 2021) Rapando, Wambani Japheth; James Ogutu; Margaret Muturi; Mathew Piero Ngugi
Staphylococcus aureus (S. aureus), Methicillin resistant S. aureus (MRSA), Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli) are common bacterial pathogens. Multi-drug resistant strains of these pathogens have led to a demand for new treatment options. O. basilicum is an important medicinal plant and has traditionally been shown to have antimcrobial values. However, there was need to validate its safety and immune modulation potential. The aim of this study was to determine the in vivo immunomodulatory, safety and in vitro antibacterial effects of O. basilicum. This was a laboratory-based experimental study. Ocimum basilicum was obtained from Siakago, Embu County. Different concentrations of the ethyl acetate leaf extract were used for testing antibacterial efficacies by the disc diffusion method followed by determination of MIC and MBC by broth dilution. Phytochemical and elemental properties of the extract were assayed by GC-MS and atomic absorbance spectrophotometry. For immune modulation, 300, 200 and 100 mg/kgbw extract concentrations were used while RRBC‘s were used as the antigen. Administration of mice with 300, 548 and 1000 mg/kgbw for 28 days was done to determine their body weight, organ weight, biochemical and hematological profiles for safety evaluation. Ethical approval for the use of mice was sought from the Kenyatta University animal care and use committee. Phytochemical composition of the extract included terpenoids, alkaloids, essential oils, fatty acids, flavonoids and aldehydes. Of the mineral elements detected in the extract, Mg had the highest concentration (1241.6±0.42 mg/100g) while cobalt had the lowest concentration (0.08±0.02 mg/100g). Heavy metals were not detected. A broad spectrum antibacterial activity was exhibited on the tested bacterial strains. The highest activity was on P. aeruginosa (27.00±2.00mm) while the lowest activity was on the isolate of S. aureus (17.33+0.58mm). The extract reversed the effects of cyclophosphamide on the myeloid stem cells by restoring the counts of Hb, WBC and RBC‘s in the blood. There was a dose dependent effect on the humoral antibody responses with the 300 mg/kgbw concentration of the extract exhibiting statisticaslly significant (12.4±0.894) antibody responses on the RRBC‘s. The extract exhibited an effect on neutrophil adhesion and a dose dependent effect on phagocytic index which wasn‘t significantly different among the extract concentrations. In DTH models, the different extract concentrations had dose dependent effects on TLC‘s and PLT counts. In vivo safety tests revealed that the 1000 mg/kgbw concentration significantly lowered PLT counts (556.00±76.00 10^3/μl) while having no effect on other blood indices. The extract was also proven safe on the liver and kidney. The ethyl acetate leaf extract of O. basilicum had antibacterial, immune modulation effects and was safe for in vivo use, therefore, this plant could be harnessed in efforts to come up with alternative and complementary therapies.
Analysis of manual liquid-based cytology, histopathology and HPV DNA testing among HIV positive women at Machakos level 5 hospital, Machakos County, Kenya
(Kenyatta University, 2022) Mutuku, Onesmus Muia; Scholastica Mathenge; Mutinda C. Kyama; Wachuka Njoroge
Liquid-based cytology is a technique of preparing a monolayer of cells by washing the cells in a phial of liquid fixative and spreading a thin layer of the sample on a glass slide. This enables a better morphological assessment of the cells, thus increasing the detection rate of cytological abnormalities. In the developed countries, the liquid based cytology technique is fully automated; however, in developing countries, the approach is undermined by its unavailability and related costs. Although conventional Pap smear examination is the primary approach for early detection of cervical cancer, it has shortcomings such as presence of obscuring materials like blood, mucus, and inflammation; hence, the sensitivity is reduced considerably. Contrary, manual liquid based cytology technique increases specimen adequacy rate and improves precursor lesions and cervical cancer detection by its ability to overcome the shortcomings experienced in Conventional Pap Smear. The main objective of this research was to compare the performance of manual liquid based technique versus that of histopathology and assess the utility of its remnant samples in detection of high risk Human Papilloma Virus. Through the use of a Manual liquid-based cytology, a prospective study of 400 cases was evaluated for pre-cancerous lesions and cervical cancer at Machakos County Hospital among women attending the Comprehensive Care Centre. Purposive sampling method was applied to obtain the study population. The principal investigator screened all the Pap smears and a pathologist reviewed all the abnormal smears. All participants with features of high-grade squamous intraepithelial lesions and above based on cytological results were referred for histopathology. In all remnant samples with abnormal lesions encompassing atypical squamous cells of unknown significance and above, detection of Human Papilloma Virus 16 and 18 was done using a real time Polymerase Chain Reaction. Chi-Square test was used to relate the groups’ nominal categorical data appropriately. Cohen Kappa test was performed to establish the exact level of agreement among HPV DNA testing, histopathology, and Manual liquid based cytology. The prevalence of cervical lesions in this study was 7.8%. There were 41 (10.3%) cases of bacterial vaginosis recorded in this study. Out of the 25 samples tested for High-risk HPV DNA, 18 (72%) were positive while 7 (28%) were negative. Ten women were referred for biopsy and histopathological examination. Of those 10, 4 (40%) had CIN II, 3 (30%) had CIN III, 2 (20%) showed features of squamous cell carcinoma while 1 (10%) was found to have chronic cervicitis. There was moderate agreement between histopathology and high risk HPV DNA testing; k=0.574, (95% CI, .41 to .60), p =0.11. There was substantial agreement between histopathology and high risk HPV DNA testing; k=0.615, (95% CI, .61 to .80), p =0.035. Overall, manual liquid based cytology was found to have a moderate level of agreement with histopathology with ability to preserve remnant samples for adjunctive tests such as HPV DNA detection; therefore, it can be considered a substitute screening method in limited resource settings.

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