MST-Department of Medical Laboratory Sciences

Plasma interferon-gamma, interleukin-10 and adiponectin levels in HIV-1 and tuberculosis coinfected injection drug users at Bomu Hospital, Mombasa, Kenya

Wed, 01 Jun 2016 00:00:00 GMT

Plasma interferon-gamma, interleukin-10 and adiponectin levels in HIV-1 and tuberculosis coinfected injection drug users at Bomu Hospital, Mombasa, Kenya Kiboi, Nathan Sub-Saharan Africa accounts for high tuberculosis cases that result from widespread HIV infections, which is exacerbated by injection substance use. Immunologically, HIV critically impairs cell-mediated host responses to Mycobacterium tuberculosis. IFN-γ, IL-10 and Acrp30 are key mediators of systemic inflammation. Although circulating IFN- and IL-10 levels are increased, Acrp30 levels are lowered and associated with disease severity among HIV and TB co-infected non-susbstance users. In contrast, circulating IFN- and Acrp30 levels are decreased while IL-10 levels are upregulated among injecting heroin addicts. However, no studies to date have reported on these cytokine profiles among Kenyan HIV-1 and TB co-infected injection drug users. This study, therefore, investigated plasma IFN-γ, IL-10 and Acrp30 levels among IDUs, and their association with CD4+ T cell counts, HIV-1 viral load and BMI. A cross-sectional study was conducted from August, 2012-November, 2013 using 138 participants recruited at Bomu hospital; a major centre for rehabilitation of drug and substance users in Mombasa County. Following informed consent, IDUs were enrolled through respondent driven sampling, snowball and makeshift methods while convenience and purposive sampling were used for recruiting the control group. IDUs and controls were screened for HIV and TB respectively through Determine™ and Bioline™ rapid tests, and Ziehl Neelsen stained sputum smears. Subsequently, the study participants were categorised into: HIV-1/TB coinfected ART-naive (n=9) and -experienced (n=27); HIV-1 mono-infected ARTnaive (n=26) and -experienced (n=13); TB mono-infected (n=21), HIV-1 negative and TB uninfected (n=25) IDUs and controls (n=17). Demographic, drug use information and physical measurements were recorded using assisted interviews. EDTA venous blood samples were collected and used for preparing plasma and enumerating CD4+ T cell counts. Frozen plasma samples were used for determining cytokine concentrations, and HIV-1 viral load. CD4+ T cell counts were enumerated using flow cytometry; cytokine levels were measured using a sandwich ELISA technique, while HIV-1 viral load was determined by RT-PCR, respectively. Across-group comparisons in continuous data were performed using Kruskal Wallis followed by post-hoc Dunn’s tests. Plasma IFN-γ (P<0.0001), IL- 10 (P<0.0001) and Acrp30 (P=0.006) levels differed significantly across groups. IFN-γ levels were high in co-infected ART-naive (P<0.001) and -experienced (P<0.001), and HIV-1 mono-infected ART-experienced (P<0.001) IDUs relative to healthy controls. IL-10 levels were elevated in uninfected IDUs (P<0.001) compared to healthy controls. Acrp30 levels were lower in TB mono-infected (P<0.01) relative to controls. IFN-γ/IL-10 ratio varied across-groups (P<0.0001) and higher in co-infected ART-naive (P<0.001) and -experienced (P<0.001), and HIV-1 mono-infected ART-experienced (P<0.001) compared to uninfected IDUs. The IFN-γ/Acrp30 ratio also differed across groups (P<0.0001) with HIV-1 mono-infected ART-experienced (P<0.001), and co-infected ART-naive xii (P<0.001) and -experienced (P<0.001) IDUs exhibiting higher ratio relative to uninfected IDUs. CD4+ T cells correlated inversely with Acrp30 (=-0.717, P=0.030) levels in TB mono-infected IDUs whereas BMI correlated positively with Acrp30 (=0.523, P=0.022) among co-infected ART-naive IDUs, respectively. Altogether, circulating IFN-, IL-10 and Acrp30 production is altered in ART-naive and -experienced HIV-1 and TB co-infected IDUs, suggesting a role as disease markers in HIV and TB co-infection among IDUs. A Thesis Submitted in Partial Fulfilment of the Requirements for the Award of the Degree of Master of Science (Medical Biochemistry) in the School of Pure and Applied Sciences, Kenyatta University. June, 2016

Prevalence of HIV/HCV co-infection amongst HIV serodiscordant couples in Thika, Kenya

Fri, 01 Apr 2016 00:00:00 GMT

Prevalence of HIV/HCV co-infection amongst HIV serodiscordant couples in Thika, Kenya Waweru, Susan Wairimu Immune defects caused by Human immunodeficiency virus (HIV) and Hepatitis C virus (HCV) co-infection could alter the course of secondary infection and dysregulated innate immune responses could contribute to a more rapid disease progression. The prevalence of HCV and HIV co-infection in Kenya varies with different risk groups. Currently, there is parity of data on the prevalence of HIV/HCV co-infection in Kenya. It is also not known how the CD4+ and CD8+ counts correlate with HIV viral load in HIV/HCV co- infected individuals. This study focused on determining the prevalence of HCV and how the CD4+ and CD8+ counts correlate with HIV viral load in HIV/HCV co-infected individuals amongst HIV serodiscordant couples in Thika. Plasma samples from HIV serodiscordant couples that had been collected over a period of 2 years (2006-2008) and stored at -20° C at the Clinical Trials Research Laboratory (CTRL) were used. All the samples from both HIV negative and positive participants were tested for HCV antibody using Murex anti-HCV (version 4.0) microelisa kit. HIV RNA viral load and CD4+/CD8+ counts were obtained from already archived data for the participants that were co-infected with HIV and HCV. A total of 385 samples from HIV serodiscordant couples were used in this study; 196 HIV positive samples and 189 HIV negative samples. Data was analyzed using mean (standard deviations), medians (inter-quartile range), Pearson correlation, Pearson chi square test, McNemar chi square test, ranksum test and t-test as appropriate using Stata version 12.1. Results obtained from this study showed that the mean age of the participants was 34.2 years. 192 of the participants were male (49.9%) and 193 (50.1%) were female. Overall, 13 (3.4%) of the participants were positive for HCV, most of whom 11(5.6%) were HIV positive, p= 0.013. There was no statistically significant difference between HCV infection on the basis of gender, 7 (3.6%) vs. 6 (3.1%), p= 0.785. A negative correlation was observed between HIV Viral load and CD4+ T cells amongst HIV/HCV co-infected participants without statistical significance; r= 0.600, p= 0.070. A positive correlation was observed between HIV Viral load and CD8+ T cells amongst HIV/HCV co-infected participants without statistical significance r= 0.4525, p= 0.162. There was a statistically significant higher median (IQR) HIV Viral load (copies/mL) at baseline among those positive for HIV/HCV co-infection than those who were HIV mono-infected, 89775 vs. 10695, P= 0.0436. Those positive for HIV/HCV co-infection had a lower median CD4+ (IQR) than those who were HIV mono-infected, however without statistical significance, 383 vs. 499, p=0.0772. Those positive for HIV/HCV co-infection had a higher median CD8+ (IQR) than those who are negative for HIV/HCV co-infection, however without statistical significance, p=0.2250. None of the serodiscordant couples were both found to be positive for HCV indicating that HCV may not have been transmitted sexually. A longitudinal study is recommended using a higher number of participants and also inclusion of more laboratory tests and parameters to give a better understanding of the effect of HIV/HCV co- infection on disease progression amongst participants. A thesis submitted in partial fulfilment of the requirement for the award of the degree of Master of Science in infectious diseases (Immunology) in the School of Medicine, Kenyatta University. April 2016

Evaluation of safety and therapeutic potential of environmental waste water lytic bacteriophage against multidrug resistant staphylococcus aureus (MDRSA) in BALB/c mice

Fri, 01 Apr 2016 00:00:00 GMT

Evaluation of safety and therapeutic potential of environmental waste water lytic bacteriophage against multidrug resistant staphylococcus aureus (MDRSA) in BALB/c mice Oduor, Joseph Michael O. Methicillin resistant Staphylococcus aureus (MRSA) pose a great threat to the global public health. Control of these bacteria has become difficult due to acquisition of resistance against even some of the best antibiotics. Thus, phage therapy could be the better alternative as they are easy to isolate and produce in mass within a short time. However, phage therapy has been a subject of debate over the years but recently there has been a renewed interest due to their proved therapeutic potential and have, therefore, found commercial application in some countries. Thus, this study was specifically designed to evaluate the efficacy of phages against MRSA both in vitro and in vivo. A litre of environmental waste water and sewage samples were collected around the county of Nairobi. The MRSA isolates were obtained from environmental waste water and sewage samples from Nairobi and its environs and evaluated for drug resistance using antibiogram test. In addition, lytic phages were isolated from these samples too. Thereafter, the in vitro efficacy of the phages against MRSA was done by spot assay and tube culture tests. Only the most virulent phage isolate was used for in vivo efficacy study which involved six groups of mice of n=5 per group (BALB/c mice; both sex). The first three groups acted as controls (group 1=only physiological saline, group 2=MRSA bacteria only and group 3=phage only) while the remaining groups were used for efficacy studies using a dosage of 108 CFU/ml for MRSA bacteria, 108 PFU/ml for phage and clindamycin at 8mg/kg. The efficacy study groups (groups 4-6) were first infected by MRSA and observed for 3 days before treatment with either antibiotic (group 4), or phage (group 5) or phage + antibiotic (cocktail therapy group 6). The mice were then observed for an additional 7 days. During the entire 10 days of observation blood samples were collected daily for bacteremia level determination before being euthanized. Different organs including the liver, brain, kidney and lungs were harvested for histopathological studies. All studies were done in accordance with the IPR Institutional Ethical Committee approved protocols. Antibiogram test indicated that MRSA isolated was a multidrug resistant strain (Figure 1). While the in vitro test showed the virulence of the phage isolates after 24 hrs culture at 370C. Toxicity test showed that phages were safe. There was no significant difference in survival rates between phage infected group and non-infected control group (p>0.05). Bacteremia was significantly lower in phage treated group as compared to other treatment groups and bacteria non-treated group (p<0.001) (Figure 2). In addition, pathological results show that phage prevented organ damage by the bacteria (Figure 3). Thus, a single dose of phage was more effective than other therapeutic agents used in the study. Results of this study show that phage therapy is safe and its application should be considered for the treatment of multidrug resistant bacterial infections. A thesis submitted in partial fulfillment for the award of the Degree of Master of Science in Infectious Diseases (Virology) of Kenyatta University

Isolation and characterization of antibiotic producing thermophilic bacillus in selected hot-springs along lake Bogoria, Kenya

Thu, 01 Jan 2015 00:00:00 GMT

Isolation and characterization of antibiotic producing thermophilic bacillus in selected hot-springs along lake Bogoria, Kenya Torome, Tom Kintet Extreme habitats are increasingly being recognized as sources of secondary metabolites which provide an encouraging source for development of novel natural pharmaceuticals. Saline and hot water lakes are now becoming appreciated as rich and untapped reservoirs of extremophilic microorganisms with previously uncharacterized functions. Bacilli, a large homogeneous group of bacteria that survive in a wide range of environmental conditions, is one such microorganism. Formation of resistant spores allows it to survive in high temperatures zones where other organisms cannot. The objective of this study was to isolate, identify, and characterize Bacillus organisms with the potential to produce secondary metabolites with antibacterial properties from the hot springs of Lake Bogoria. Eighty samples were collected from surface and sediment waters of selected hot springs and inoculated directly into nutrient broth in universal bottles. Samples were initially incubated at 45oC for 48 hours and growth was examined by checking for turbidity. Out of the 80 bottles inoculated with sample, thirty three exhibited growth. Subsequent streaking on nutrient agar and Gram staining showed that nine of the isolated organisms were Gram-positive rods, twenty were Gram-negative rods, and four were Gram-positive cocci. Only Gram-positive rods were processed further. Biochemical characterization revealed that all nine processed isolates were catalase-positive and non produced gas upon sugar fermentation. They however had varying results for indole, motility, lactose fermentation, glucose fermentation and H2S production. Antibacterial profiling was performed using ATCC organisms: Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, Escherichia coli 35218, and Pseudomonas aeruginosa ATCC 27853. Isolates D1, D5, D8, D22 and S15 inhibited growth of Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 which was shown by clear zones of inhibition around the discs. No inhibition was noted against S. aureus ATCC 29213 and E. coli 35218. Phylogenetic analysis of amplified 16S rDNA gene showed that D1, D5, D8, D22 and S15 formed close phylogenetic clusters with known members of Bacillus organisms with a (88-99%) sequence identity. The current study shows the presence of thermophilic Bacillus species, which are producers of biomolecules with antibacterial properties within the hot springs of Lake Bogoria. Characterization of the antibacterial compounds produced will be useful for the discovery of novel antimicrobial substances that are effective against wide range of pathogens. A thesis submitted in partial fulfilment of requirements for the award of the degree of Master of Science (biotechnology) of Kenyatta University, 2015