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Item Aflatoxins in peanuts and the prevalence of aflatoxin induced hepatocellular carcinoma in Busia and Kisii Central Districts, Kenya(Kenyatta University, 2015-12) Chengo, Nelson MenzaAflatoxin is a carcinogenic toxin produced mainly by Aspergillus jlavus and contaminates foods including peanuts. Aflatoxin is associated with liver failure, hepatocellular carcinoma (HCC) and death. Many people are exposed to chronic levels of aflatoxins through consumption of contaminated foods. In Kenya, most efforts have been focused on aflatoxin in maize while other highly predisposed foods such as peanuts have received little attention. Also limited studies have been done to link aflatoxin to HCC. This study identified aflatoxin producing Aspergillus species and the type and levels of aflatoxin contamination of various varieties of peanuts (Arachis hypogaea L.) in Busia and Kisii Central districts. It also determined the peanut producers' exposure to aflatoxins and the prevalence of aflatoxin induced HCC among patients from the study districts. Cross-sectional and retrospective study designs with systematic random sampling technique were adopted. One hundred and two (102) peanut and urine samples were collected from peanut growers in each district and transported to Cooppers Labs, Nairobi for analysis. Aspergillus species' were identified using plate technique of serially diluted samples on modified Rose Bengal agar. Types and levels of aflatoxins were analyzed using high performance liquid chromatography (HPLC) technique while aflatoxin B (AFB) gual in urine was determined using fluorescence Spectrophotometer. Analysis of records for patients from Busia and Kisii Central districts who attended Moi Teaching and Referral Hospital in January 2010 to December 2012 was done to determine the prevalence of HCC among the study population. The diagnosis of HCC was confirmed by the presence of AFB 1 guanine adducts in urine or AFB 1 albumin adduct in blood. The levels of total aflatoxin ranges were 0.1 to 2681lg/kg and 1.63 to 591.11lg/kg in peanuts from Busia and Kisii Central respectively. Majority of peanuts samples had levels within Kenya Bureau of Standards (KEBS) and European Union (EU) regulatory limits for total aflatoxins. Aflatoxin type Bl was the most dominant (t = 12.4, df= 3, P = 0.034). Overall, the occurrence of Aspergillus jlavus L strain and A. flavus S strain were significantly higher than other species identified (H = 15.55, df= 4, P = 0.004) in peanuts from the two districts. However, A. jlavus S-strain was the most dominant species (F=3.15, df=25, P=0.031) with an overall mean occurrence of 45.1%. Oil content in peanuts decreased with an increase in aflatoxin levels (r = -0.496, P = 0.031) except in peanuts of Uganda local red variety from Kisii Central. Overall, males in both districts had slightly higher incidences (9.8%) of exposure to urinary Aflatoxin B than females at 5.4%. The prevalence of aflatoxin induced HCC among the patients was 19.73%. There is need for urgent awareness through campaign among the peanuts producers on the aflatoxin levels and promote sound practices when handling peanuts. Continuous screening of patients with liver disorders should be done for early detection of HCC. The high levels of aflatoxins in peanuts suggest the necessity to screen other foods consumed in the study areas. The findings will form basis of policy development for aflatoxin contamination control and management of aflatoxin induced HCC in Kenya.Item Analyses of class and subclass antibody of circulating immune complexes in children with severe Plasmodium Falciparummalaria in endemic regions of Western Kenya(Kenyatta University, 2010-06) Mibei, Erick KipsangPlasmodium falciparum infection is characterized by deadly complications such as severe malaria-associated anaemia (SMA) and cerebral malaria (CM).The exact mechanisms underlying pathogenesis of these severe forms of Plasmodium falciparum malaria are not fully understood yet they are associated with a lot of morbidity and mortalitv. Studies have shown a link between severe P. falciparum malaria and levels of circulating immune complexes (CIC) but the exact role of these CICs in the pathogenesis of severe P. falciparum malaria is still unclear. This study aimed to investigate the quantitative and qualitative differences in antibody classes and subclasses in serum immune complexes (lCs) between children with the severe forms of P. falciparum malaria and those with uncomplicated malaria as well as identifying the predominant P. falciparum antigens that contribute to IC formation in these clinical groups. A total of 75 children with SMA and 32 children with CM were enrolled from hospitals in western Kenya and matched with 74 and 52 control children respectively with uncomplicated symptomatic malaria. IC levels were measured using solid phase ELISA protocols and antibody classes and subclasses were identified using polyspecific sera for the classes or monoclonal antibodies for the subclasses. ICs were purified using polyethylene glycol (pEG) precipitation. The isolated ICs were dissociated by using an acidic buffer (Glycine- HCL pH 2.0). These were then electrophoresed on one-dimensional and two-dimensional polyacrylamide gel blotted by Western transfer and probed using human anti-F. falciparum antibodies. The study showed a general increase in levels of ICs as a result of P. falciparum infection in severe malaria cases and their symptomatic controls. Although IgG IC levels were elevated in children with severe malaria upon enrolment, children with CM had the highest levels of ICs for all the antibody classes. Conditional logistic regression showed a borderline association between IgG4-containing ICs and increased risk of SMA (OR = 3.11, 95% Cl 1.01 to 9.56, P = 0.05). Total IgG-containing ICs (OR = 2.58, 95% Cl 1.20 to 5.53, P < 0.(2) and IgE-containing ICs (OR = 3.27, OR 1.38 to 7.78, P < 0.01) were associated with increased risk of CM. Six specific P. falciparum antigens were found to be associated with severe malarial anaemia while another three antigens were associated with cerebral malaria when compared to their specific controls. While when SA and CM where compared together, a 91Kda antigen was highly associated with SA (P < 0.01), while a slightly lighter antigen of about 87 Kda was significantly associated with CM (P < 0.01). These findings have demonstrated quantitative and qualitative differences in ICs in children with SMA and CM and this underscores the potential mechanisms of the pathophysiology of the disease. Furthemore the findings of this study suggested having higher IgG4-containing ICs is a risk factor for SMA while higher IgG and also IgE-containing ICs are both associated with CM pathology. This suggest that although SMA and CM were characterized by high levels of ICs, the class and subclass make up of these KCsas well as the role that they play in each may be distinct. This study demonstrated an association between malaria antigens and severity of the disease hence there is need for full characterization of the parasite antigens. These findings may contribute to a better understanding of the role of different antibody classes and subclasses in protective or damaging mechanisms and may provide new insights into development of effective malaria control strategies and vaccine development.Item Human Papillomavirus Prevalence, Genotypes, and Factors Associated with Cervical Abnormalities Among HIV-Positive and Negative Women in Eastern and Central Regions, Kenya(2022) Njue, James Kinoti; Margaret Muturi; Lucy KamauHuman Papillomavirus (HPV) associated cervical cancer caused 3,286 deaths in 2019 in Kenya with a national cervical screening rate of only 3.2%. The overwhelming predominance of distinctive HPV genotypes infection has implications regarding the HPV vaccination efficacy. Approaches to reduce cervical cancer incidence and mortality by either ‘screen-and-treat” or “screen, triage and treat” are based on the diagnostic accuracy of available screening methods as well as awareness of the disease and its signs and preventive measures. This comparative study aimed to investigate the Human Papillomavirus prevalence, genotypes, and factors associated with cervical neoplasia among HIV-infected and non-infected women. The diagnostic accuracy of cervical screening methods was also determined. HIV-infected (cases) and non-infected (control) women aged 18-46 years women underwent cervical screening and colposcopy-biopsy confirmatory test alongside filling out a questionnaire on awareness of cervical cancer and its risk factors. A cervical broom was softly rotated 360 degrees five times to exfoliate cells from the region of the transformation zone, squamocolumnar junction, and endocervical canal for HPV genotyping, Pap smear followed by VIA/VILLI test. Laboratory outcome and questionnaire data statistical relationships were computed using the Pearson chi-square test. 161(50.8% cases and 156(49.2%) control, mean age: 34.3, SD ±10:4, range 18-46 years were recruited from Embu (85/317(26.8%)), Isiolo (64/317(20.2%)), Kirinyaga (56/317(17.7%)), Meru (81/317(25.6%)), and Tharaka-Nithi (31/317(9.8%)) Counties. 81/317(25.6%), 84/317(26.5%), 96/317(30.2%) and 78/122(63.9%) participants had VIA, HPV DNA-PCR, Pap smear and cervical histology positive results respectively. A higher primary diagnostic accuracy was established by HPV DNA-PCR (sensitivity: 95.5%; specificity: 92.6%) than Pap smear and VIA test while in triage testing, high sensitivity was obtained by HPV DNA-PCR parallel testing with VIA test (92.6%) and Pap smear test (92.7%). HPV genotypes distribution by cervical dysplasia were CIN1 (cases: HPV81 (12/317(3.8%)) and HPV11 (2/317(0.6%)); control: HPV53 and 66 co-infections (1/317 (0.3%)), CIN2 (cases: HPV11, HPV16, HPV66 ((1/317 (0.3%) each), HPV81 (6/317 (1.9%)); control: HPV81 (2/317(0.6%)) and Invasive Cervical Carcinoma (cases: HPV16 (1/317(0.3%)) and HPV81 (3/317(0.9%)). Most sample isolates had phylogenetic relationship with reference sequences from Iran [22.3% (17/76)], Kenya [22.3% (17/76)], Bangkok [14.4% (11/76] and India [7.9% (6/76)]. Knowledge of HPV screening was significantly influenced by residence, age, education level, married-marital status, religion, and hormonal contraceptive use (p<0.005). Fear of embarrassment was the main reason for failing to undergo cervical screening. HPV-PCR is recommended for routine testing and evaluation of Pap test results or during the treatment of cervical lesions. There was a higher frequency of both high-risk and low-risk HPV genotypes and the existence of non-vaccine HPV genotypes associated with cervical dysplasia among HIV-infected than HIV-uninfected women. Increased knowledge, willingness, and perceiving cervical screening as important, as well as personal acceptability to vaccinate against HPV, may reduce the cervical cancer burden. Keywords: Human Papillomavirus, cervical cancer, screening, genotyping, Knowledge, Attitude, Practice and Perception, HPV vaccinationItem Screening of Filovirus Ribonucleic Acid in Humans, Wild Caught Non-Human Primates, Bats and Rodents in Laikipia North Sub-County, Kenya.(Kenyatta University, 2022) Auma, Ambala Peris; George Gachara; Nelson Menza; Joseph KamauIn the recent decade, highly pathogenic human viruses have constantly emerged or re-emerged in different geographical locations worldwide approximately annually with the majority jumping from animals to humans. The major factors that have led to emergence of these viral diseases include urbanization, changes to local ecosystem and changes in human behaviours. Many of the emerging and re-emerging viruses are Ribonucleic acid (RNA) viruses with a high potential for re-assortment, recombination and mutation leading to new/different pathogenic viral strains. Despite the fact that non-human primates, bats and rodents have increasingly been implicated as potential sources of emerging zoonotic viral diseases, there is paucity of scientific data on Filoviruses circulating at the animal human interface in Laikipia North Sub-county, Kenya. The aim of this study was to detect and characterize filoviruses circulating in Laikipia, County Kenya. A cross sectional study design was adopted. Sampling technique employed in this study for human population was convenience sampling while for animal samples, capture release method using PREDICT 2 protocols was used. A total of 1092 samples of whole blood/blood swabs/blood clots (n=377), oral swabs (n=405) and rectal swabs/fecal (n=310) material were obtained from consenting individuals and the aforementioned animals. The samples were stored in TRizol® reagent (Thermo Fisher Scientific). The viral ribonucleic acid (RNA) was extracted. From the RNA, complimentary deoxyribonucleic acid (cDNA) was synthesized using Invitrogen superscript III first strand synthesis kit. The cDNA was amplified using PREDICT 2 filovirus universal primers in a consensus reverse transcriptase polymerase chain reaction (RT-PCR) targeting the Large (L) gene. In addition, immunoglobulin gamma enzyme linked immunosorbent assay (IgG ELISA) was conducted only on human and non-human primate serum samples. The IgG ELISA targeted IgG antibodies against Ebolaviruses and Marburg virus. Risk factors associated with zoonotic infections were assessed using surveys. All the samples tested negative for filoviruses using consensus RT-PCR and IgG ELISA. On risk factors analysis, bush meat was reported to be in circulation while less than 10% of the participants reported having interaction with wild animals in the last 6 months of the study. Despite the negative results in laboratory assays (consensus RT-PCR and IgG ELISA),cultural beliefs and practices, the circulation of bush meat and interaction of the community with wild life pre-disposes these population to the risk of contracting zoonotic viruses other than filoviruses. This study has identified potential risk factors associated with zoonotic disease transmission at the study area which are getting into contact with animals, sharing of water points with animals and use of untreated water for domestic use. These results suggest that circulation of filovirus was absent in humans, non-human primates, rodents and bats from Laikipia North Sub-county. Considering our findings, the study recommends continuous surveillance, systematic collection of samples from human population and animals for laboratory analysis for planning, implementation and evaluation of potential emerging filoviruses within the region and the use of both serological and molecular assays for detection of filoviruses.Item Spatial and temporal distribution of Aedes mosquitoes, Dengue and Chikungunya Viruses and their Phylogeny along the Coastline of Kenya(Kenyatta University, 2020-07) Ngala, Chome JonathanThere are arthropod-borne disease outbreaks as a result of pathogen influx including arboviruses which are transmitted by strains of Aedes species that occur periodically in varying spots in Kenya. However, there has been paucity of documented information on the epidemiology of Aedes mosquitoes involved in transmission of different strains of viruses. This cross sectional study determined spatial and temporal distribution of Aedes mosquitoes, Dengue and Chikungunya viruses, and their phylogeny and vector-virus co-infections during dry and wet seasons. Indoor and outdoor sampling of adults Aedes mosquitoes was done using Biogent Sentinel trap baited with solid carbon dioxide and Prokopack aspiration technique. Aedes mosquitoes were identified and sorted according to collection site, sex, physiological status and species using their morphological features and molecular techniques. Sentinel sites coordinates were recorded by Global Positioning System receiver with spatial and temporal maps generated using ArcGeographical information system. RNA was extracted from Aedes mosquitoes using Trizol®. Identification of Aedes species, Dengue and Chikungunya was done using Polymerase Chain Reaction. Sequencing of amplicons was done using Sanger high-throughput technique and their proportions analysed by R-statistics. Phylogeny tree files were generated using Randomised Accelerated Maximum Likelihood and trees plotted using interactive tree of life. A total of 37,220 Aedes mosquitoes belonging to eight species were collected and grouped in pools of 20 mosquitoes. Aedes aegypti formosus was dominant at 62.5%. Aedes aegypti aegypti was identified for the first time along the Coastline of Kenya. There was no effect of season on the distribution and proportion of Aedes species along the Coastline. Aedes mosquitoes belonged to the upper clade of the phylogenetic tree. Four serotypes of Dengue virus were identified with DENV-4 identified for the first time in Aedes mosquitoes in the region. Only the East/Central/South African (ECSA) genotype of Chikungunya virus was isolated and seasons did not influence the distribution of both viruses along the Coastline (p>0.001). Aedes mosquitoes were closely related to previous isolates and to those from Uganda, Senegal and Thailand. DENV-1 isolates were closely related to those from India, DENV-2 isolates were closely related to those from Pakistan, and DENV-3 isolates were closely related to those from Brazil while DENV-4 isolates were closely related to those from Haiti. Chikungunya ECSA genotype isolates were closely related to previous Kenyan isolates and to those from South Africa and Tanzania. There were co-infections of Dengue and Chikungunya viruses in Aedes mosquitoes. Aedes aegypti s.l and Aedes pembaensis had co-infections of all viruses. Prevalence of Dengue virus was at 7.9% while Chikungunya was at 2.1%. These results are important as they give information on areas of high risk for the virus outbreaks. Surveillance of entomological infection by viruses and implementation of their appropriate control measures should be taken by the Ministry of Health.Item Streptococcus Pneumoniae Serotype Prevalence, Antibiotic Susceptibility and Associated Risk Factors among Children Attending Gertrudes Children’s Hospital in Nairobi City County-Kenya(Kenyatta University, 2020-06) Nyongesa, Walekhwa MichaelPneumococcal disease remains the biggest killer of children living in Kenya. This is true despite inclusion of the 10-valent pneumococcal conjugate vaccine in the Kenya Expanded Program on Immunization. Serotype replacement, emergence of antibiotic resistance, inaccurate laboratory diagnosis due to optochin resistant bacterial types and a range of environmental and host related risk factors have been touted to be the cause of these statistics elsewhere. This study sought to establish prevalence of Streptococcus pneumoniae serotypes, antibiotic susceptibility patterns and associated risk factors among PCV-10 vaccinated and unvaccinated children attending Gertrude’s Childrens Hospital. A total of 206 children were recruited for this study. Nasopharyngeal swabs were the main specimen used. Culturing and isolation of the bacteria was done on blood agar with gentamicin and plain blood agar plates respectively. Optochin and bile solubility (where necessary) tests were done as confirmatory assays for the bacteria. Pneumococci serotyping was done using the Gold Standard Quellung Reaction test while the disk diffusion method was used to asses antibiotic susceptibility profiles. Out of the 206 subjects sampled, 20.39% (n=42) were found to be carriers of the bacteria. About 52% (n=22) of the carriers had received the recommended dose of PCV-10, while 48% (n=20) had not. Almost all (n=41; 19.90% of subjects) isolates contained non-vaccine type serotypes, while n=1 of the isolates (0.49% of subjects) were both optochin resistant and untypeable. Serotypes 28F, 6A, 11A, 3 and 7C were prevalent in both vaccinated and unvaccinated children, whereas serotypes 23A, 17F, 35F, 48, 13 and 35B, and 23B, 20, 19B, 21, untypeable, 15B and 39 were found among unvaccinated and vaccinated cohorts, respectively. Thirty nine (92.86%) of pneumococci isolates were susceptible to erythromycin, 39 (92.86%) were susceptible to vancomycin, 8 (19.86%) were susceptible to oxacillin; 40 (95.24%) were susceptible to clindamycin, 24 (57.86%) were susceptible to ceftriaxone while 18 (42.86%) were non-susceptible. The risk of nasopharyngeal carriage decreased insignificantly when the subject was female (odds ratio [OR]: 0.766, 95% CI: 0.388, 1.511, p-value=0.442). Children between the age of 25-36 months (OR: 1.147 (95% I: 483, 2.722) and 37-48 months (OR: 1, 95% CI: 0.286, 3.501) had an insignificant elevated risk of nasopharyngeal carriage of the bacteria. Children whose mothers were non-cigarrate smokers exhibited low odds of carriage (OR: 0.764 (95% CI: 0.077, 7.537; p=0.818). Serotype replacement, resistance to penicillins and exposure to smoke were correlated with incresaed risk of nasopharyngeal carriage. Continuous and broader epidemiological surveys should be carried out in the entire country to accurately determine the degree of serotype replacement and; people should be sensitised on judicious use and/or consumption of antibiotics. Optochin test should be introduced as a routine assay in diagnosis of Streptococcus pneumoniae in hospitals.Item Total blood volume, iron status and hematological profiles of whole blood donors at Kenyatta National Hospital, Nairobi City County, Kenya(Kenyatta University, 2023) Njenga, John K.; Scholastica Mathenge; Nelson Menza; Jessie GithangaThe effectiveness of blood donation exercises relies on safeguarding prospective blood donors’ and recipients’ health. This may be realized through collection of safe and appropriate blood volume. An ineffective donor selection criterion may expose potential donors to vasovagal reactions and lead to collection of blood with hematological abnormalities. In Kenya, there is paucity of data on donors’ total blood volume (TBV), iron status, hematological profiles and deferral rates. The objectives of this study were to determine: the percentage of blood volume donation, reliability of using Nadler’s or Lemmens-Bernstein-Brodsky’s equations to calculate TBV, donor iron status, hematological profiles and deferral rates among blood donors attending Kenyatta National Hospital. A cross-sectional study design was adopted. Using a systematic random sampling technique, 384 study participants were recruited. Donors were grouped into two categories (eligible and deferred donors) based on the donor recruitment outcome. Study questionnaires were used to collect donors’ demographic information. Eligible donors were checked for the total blood volume using Nadler’s and LemmensBernstein-Brodsky’s equations. Eight (8) ml of blood samples were collected from the donated units and halved into plain tubes and EDTA tubes. Serum ferritin levels were analyzed using Biomeriex Mini Vidas® analyzer while hematological parameters were determined using HumaCount 5D® analyzer. Ethical approval was sought from KNHUoN ethical review committee. Non-parametric variables were analyzed using Mann Whitney U test and Kruskal Wallis H test. Intra–class Correlation Coefficient (ICC) was used to assess reliability of using the two total blood volume equations. This study identified 202 eligible donors, majority being male donors (173 vs. 29). Using Nadler’s and Lemmens-Bernstein-Brodsky’s equations the percentage of blood volume donated by eligible donors was 11.7% and 11.6%, respectively. Female donors donated significantly higher blood volume than their male counterparts (P=0.01). Reliability tests showed an excellent reliability of using either Nadler’s or Lemmens-Bernstein-Brodskys’ equations (average measure of 0.996 and single measure 0.991). Among eligible donors the prevalence of iron deficiency was 2.5%, whereas the prevalence of anemia was 7.4%. Female blood donors had a higher prevalence of iron deficiency (6.9% vs. 1.7%), whereas male donors had a high prevalence of anemia (7.4%vs.0%). Male donors had a significantly higher ferritin levels than female donors (P=0.01). The median counts of all eighteen hematological parameters analyzed were within local reference ranges. However, seven hematological parameters (RBC, Hgb, MCH, MCHC, monocytes, eosinophils and platelets) had significant variation from normal values (P<0.05). Additionally, male donors had significantly higher values for; red cell count, hematocrit and hemoglobin (P=0.01) whereas female donors had significantly higher lymphocyte and platelet counts (P=0.05). This study found a donor deferral rate of 47.4%. Temporarily deferred donors had a higher rate than permanent ones (93.4% vs. 6.6%). Donors with high deferral rates were first-time donors and female donors. In addition, deferral rates increased with the advancement of age. The leading causes of temporary deferrals were medication and low hemoglobin, whereas high blood pressure and diabetes were the main causes for permanent deferral. In conclusion, this study observed majority of donors met the threshold for safe blood donation. However, few donors had lower TBV, others had iron deficiency, anemia and hematological abnormalities. This study recommends a review of donor recruitment criteria to incorporate assessment of total blood volume, iron status, total blood count and advocate for blood donation awareness campaigns.