Caroline Wangari Ngugi

Antivenom administration is currently the only therapy for snakebite envenoming. However, in sub-Saharan Africa, inadequate quality control systems have led to deficits in the availability, accessibility, efficacy and safety of regionally available antivenoms, which, in turn, hinder snakebite treatment and management in the region. To address this impediment to snakebite treatment in Kenya, this study aimed to assess the preclinical neutralising potencies of four different antivenoms previously or currently available in Kenya (SAIMR polyvalent, AFRIVEN, PANAF-Premium^TM and Inoserp^TM) against key snakes of medical importance in the region, towards establishing a national antivenom quality control laboratory. Venoms were extracted from the Kenyan “big five” medically important snake species: Naja ashei, Naja pallida, Naja nigricollis, Dendroaspis polylepis and Bitis arietans, and their lethal potencies were determined using a murine median lethal dose (LD₅₀) assay. In vitro immunological assays (ELISAs and immunoblotting) and an established preclinical murine in vivo neutralisation assay (median effective dose [ED₅₀]) were used to assess the immunoglobulin-binding and venom-neutralising efficacies of the test antivenoms. In vitro assays revealed high venom-binding titres of SAIMR polyvalent, AFRIVEN and PANAF-Premium^TM, and reactivity to a wide range of venom proteins across the different snake venoms. Contrastingly, Inoserp^TM antivenom had low binding titres and poor reactivity to the snake venom proteins. These findings were aligned with the in vivo results, where SAIMR polyvalent, AFRIVEN and PANAF-Premium^TM showed potent venom-neutralising efficacies against all the tested snake venoms, while Inoserp^TM had low potency and failed to neutralise the lethal effects of N. ashei, N. pallida and D. polylepis venoms at the manufacture-claimed doses. Based on these robust preclinical results, we conclude that SAIMR polyvalent, AFRIVEN and PANAF-Premium^TM antivenoms offer considerable potential for the treatment of envenoming by diverse medically important snakes in Kenya. The observed deficiencies with the Inoserp^TM product highlight the importance of (i) robust, independent preclinical antivenom efficacy testing and (ii) the value of establishing a quality control laboratory to inform local regulatory and procurement decision making.

Papillomaviruses can infect animals and humans, causing benign lesions and cancer. Chimeric vaccines may address the limited coverage of current papillomavirus vaccines by providing cross-type immunity. The present study focused on the in silico design of a chimeric mRNA vaccine targeting a wide range of human papillomaviruses (HPV). The study incorporated 25 amino acid sequences derived from L1 and L2 capsid proteins of 14 high-risk and 11 low-risk HPV strains. These sequences underwent multiple sequence alignment, and the resulting sequences were used to develop consensus sequences. Computational approaches were then applied to predict and identify immunodominant T-cell and B-cell epitopes. The mRNA vaccine structure was designed by merging codon-optimized multi-epitope chimeric peptides with regulatory components that enhance both transcription and translation efficiency. The present study identified 19 T and 6 B cell epitopes, which were evaluated as non-toxic, non-allergenic, highly antigenic, and fully or partially conserved. The final multi-epitope peptide vaccine had a molecular weight of 60,161.29 kDa, a theoretical isoelectric point (pI) of 9.44, a solubility index of 0.451, and an antigenicity score of 0.9178. The mRNA vaccine exhibited a stable mRNA structure with a minimum free energy of -731.10 kcal/mol and an estimated molecular weight of 644.98 kDa. The proposed vaccine demonstrated no cross-affinity with the human genome and attained a worldwide population coverage rate of 86.24%. The vaccine formed a stable docking complex and exhibited strong interactions with major histocompatibility class I and class II molecules, as well as Toll-like receptor 4 (TLR4), Toll-like receptor 9 (TLR9), and the B-cell receptor. Binding affinities were assessed based on free energy (ΔG) values of -34.01 kcal/mol and -20.77 kcal/mol for major histocompatibility complex (MHC) class I and II, respectively, -1377.5 kcal/mol for TLR4, -24.19 kcal/mol for TLR9, and -34.41 kcal/mol for the B-cell receptor. This vaccine triggered both antibody and cell-mediated immune responses and increasing levels of IFN-γ and the interleukins IL-2, IL-10, and IL-12. This vaccine is considered preventive against multiple HPV infections. Nonetheless, in vitro and in vivo investigations are necessary to validate the safety and efficacy of this vaccine.

(1) Background: Human papillomaviruses (HPV) are primarily related to cervical cancer around the world. This study presented an in silico design of a chimeric L1/L2 mRNA-based vaccine candidate against a diverse spectrum of human papillomaviruses; (2) Methods: We employed a computational strategy to predict and screen for antigenicity, allergenicity, toxicity, and conservation of immunodominant cytotoxic T cells, helper T cells, and linear B-cell epitopes from the most conserved HPV L1 and L2 proteins. The selected epitope peptides were coupled together with appropriate linkers to create a multi-epitope chimeric peptides. The mRNA-based vaccine was developed by combining an optimized open reading frame with co-translation regulation and co-transcription components; (3) Results: Twenty five (25) non-toxic, non-allergenic, highly antigenic, and conserved epitopes were identified. The final vaccine had a molecular weight of 60161.29 kDa, a theoretical pI of 9.44, and a solubility of 0.451. The mRNA-based vaccine had a stable mRNA structure with a minimum free energy of -731.10 kcal/mol and an estimated molecular weight of 644.98 kDa. The suggested vaccine had no cross-affinity for the human genome and achieved a global population coverage rate of 86.24%. The vaccine demonstrated a stable docking complex and robust interactions with Toll-like receptor 4 (TLR4) with binding affinities of -1377.5 kcal/mol and was capable of stimulating immunoglobulin IgG, IgM, T and B cells lymphocytes, and cytokines by in silico simulation; (4) Conclusion: The vaccine is considered a preventive and therapeutic candidate; however, further in vitro and in vivo tests are needed to prove its safety and efficacy.

Background: Ruminant mastitis continues to be a cause of economic losses in the dairy industry and remains a major public health hazard globally.Objectives: This cross‐sectional study was carried out in Mukurweini Sub‐County of Nyeri County, Kenya, to investigate the prevalence of bacteria causing mastitis, risk factors associated with goat mastitis and the antibiotic resistance profiles of bacteria isolated from the goat milk.Methods: Farm level data on risk factors for mastitis was obtained from 56 farmers using a semi structured questionnaire. A total of 189 goat milk samples were collected. The goat's udder was observed for signs of clinical mastitis and the California Mastitis Test (CMT) used to test the milk for sub‐clinical mastitis. All samples were then cultured for morphological identification of bacteria and strain typing by Matrix Assisted Laser Desorption/Ionization (MALDI)‐Time of Flight (ToF) technique. Antimicrobial susceptibility of the isolated Staphylococcus aureus, coagulase‐negative Staphylococcus (CoNS), Escherichia coli, Klebsiella oxytoca, Pseudomonas spp., Enterobacter spp., Proteus vulgaris and Escherichia vulneris to eight commonly used antibiotics was done by the disc diffusion method and validated by determining the presence of antibiotic resistance genes (mecA and blaTEM) using polymerase chain reaction method.Results: The prevalence of clinical mastitis was 1.1% (2/189) while that of sub‐clinical mastitis was 84.7% (160/189). Higher (p < 0.05) prevalence of mastitis was observed in goats whose houses were cleaned fortnightly and in cases where farmers used same towel to dry different does’ udders during the milking process. Thirteen different bacterial species were isolated from the milk samples and identified by MALDI‐ToF, and these included S. aureus (22.0%), CoNS (20.3%), E. coli (18.1%), Pseudomonas spp. (14.3%), Enterobacter spp. (10.4%), K. oxytoca (6.0%), E. vulneris (1.7%), P. vulgaris (1.7%), Raoutella ornithinolytica (1.7%), Stenotrophomonas maltophilia (1.1%), Pantoea agglomerans (1.1%), Serratia marcescens (1.1%) and Cedeceas spp. (0.6%). One hundred pathogenic bacterial isolates were randomly selected and tested for antibiotic sensitivity to eight antibiotics out of which S. aureus were 97.5% resistant to Oxacillin and 100% sensitive to Ciprofloxacin. The CoNSs were 100% resistant to Oxacillin and 100% sensitive to Ciprofloxacin. E. coli were 93.9% resistant to Oxacillin, 69.7% sensitive to Ciprofloxacin and 87.9% sensitive to both Amoxicillin/Clavulanic acid and Meropenem. The antimicrobial resistant genes detected in S. aureus and E. coli were mecA [66.7%, 0%], and bla_TEM [20% and 78.3%], respectively.Conclusion: In conclusion, the study showed that most of the does were affected by subclinical mastitis with the main causative bacteria being Staphylococci spp. and coliforms. Farmers need to be trained on improved control of mastitis by adoption of good milking practices and use of CMT kit for early detection of mastitis. Occurrence of multidrug resistance by key mastitis causing pathogens was shown to be prevalent and therefore there is need for development of intervention strategies.

The menace of antimicrobial resistance to public health is constantly arising globally. Many pathogenic bacteria use mechanisms such as mutations and biofilm formation, which significantly reduces efficacy of antimicrobial agents. In this cross-sectional study, we aimed at determining the prevalence of selected extended spectrum β-lactamase (ESβLs) genes and analyse the possible biofilm formation abilities of the isolated bacteria causing urinary tract infection among adult patients seeking medicare at Kiambu Level 5 hospital, Kenya. The double-disk synergy test was used for phenotypic identification of ESβLs producing isolates, while microtiter plate assays with some modifications were used to test biofilm formation analysis. A total of 10 isolates were bioassayed for ESβL genes presence out of 57 bacteria isolates obtained from urine samples. From this study, the bla _TEM genes were found to be the most prevalent ESβLs genes (100%), followed by bla _OXA and bla _SHV genes at 40% and 30% respectively. In addition, the co-carriage of bla _TEM and bla _SHV was revealed at 50% lower than that of bla _TEM + bla _OXA genes at 66.7% among the study E. coli isolates. Biofilm formation finding disclosed that most of the isolates form biofilms 36 (63.2%), with Gram-negatives being the most biofilm formers 25 (69.4%) compared to the Gram-positive 11 (30.6%). E. coli 15(41.7 %), Klebsiella sp . 7(19.4%) and S. aureus 7(19.4%) were the most common biofilm formers. Further analysis showed no significant difference in biofilm formation among all tested isolates with a p-value of more than 0.05. However, overall Gram-positive isolates had a significant P-value of 0.056. Although biofilm formation’s impact on urinary tract infections is not fully recognized, the carriage of ESβLs resistance genes and the biofilm formation ability negatively impact effectiveness of UTI treatment. Therefore, we advocate for surveillance studies to map ESβLs distribution and biofilm formation genes among UTI etiological agents to halt UTI treatment failure.

Drug-target genes and their spontaneous mutations associated with resistance to first-line, second-line, third-line, novel and repurposed anti-tuberculosis drugs in Mycobacterium tuberculosis resistant strains - IJMMTD- Print ISSN No: - 2581-4753 Online ISSN No:- 2581-4761 Article DOI No:- 10.18231/j.ijmmtd.2022.040, I

Multi-drug resistant (MDR) Salmonella enterica Enteritidis is one of the major causes of foodborne illnesses worldwide. This non-typhoidal Salmonella (NTS) serovar is mainly transmitted to humans through poultry products. Bacteriophages (phages) offer an alternative to antibiotics for reducing the incidence of MDR NTS in poultry farms. Phages that survive the harsh environment of the chicken gastrointestinal tract (cGIT), which have low pH, high temperatures, and several enzymes, may have a higher therapeutic or prophylactic potential. In this study, we analysed the stability of 10 different S. Enteritidis phages isolated from Kenyan poultry farms in different pH-adjusted media, incubation temperatures, as well as simulated gastric and intestinal fluids (SGF and SIF, respectively). Furthermore, their ability to persist in water sources available in Kenya, including river, borehole, rain and tap water, was assessed. All phages were relatively stable for 12 h at pHs ranging from 5 to 9 and at temperatures ranging from 25 °C to 42 °C. At pH 3, a loss in viral titre of up to three logs was observed after 3 h of incubation. In SGF, phages were stable for 20 min, after which they started losing infectivity. Phages were relatively stable in SIF for up to 2 h. The efficacy of phages to control Salmonella growth was highly reduced in pH 2- and pH 3-adjusted media and in SGF at pH 2.5, but less affected in SIF at pH 8. River water had the most significant detrimental effect on phages, while the other tested waters had a limited impact on the phages. Our data suggest that these phages may be administered to chickens through drinking water and may survive cGIT to prevent salmonellosis in poultry.

Cervical cancer caused mainly by high risk human papillomavirus (HPV) 16 and 18 strains is the second most prevalent cancer of women in Kenya. It is often diagnosed late when treatment is difficult due to very low percentage of women attending screening thus, mortalities remain high. The most available tests in low-and-middle-income countries (LMICs) have relatively low specificity, low sensitivity, require a laboratory setting and huge technical and financial support not readily available. HPV 16/18 E6 oncoprotein has been identified as a potential biomarker in a more specific early diagnosis of cervical cancer. This retrospective cross-sectional study developed a paper-based nanokit with enhanced detection of HPV 16/18 E6 oncoprotein for cervical cancer screening. The HRP labelled antibodies HPV 16 E6/18 E6-HRP (CP15) passively conjugated to citrate stabilized 20nm gold nanoparticles were evaluated for immune sensing mechanism using a recombinant viral HPV E6 protein. The diagnostic accuracy was evaluated using 50 tissue lysates from formalin fixed paraffin embedded cervical biopsy, including control (n=10), Mild Dysplasia (n=10), Cervical intraepithelial neoplasia 3 (CIN3) (n=10), Cervical intraepithelial neoplasia 4 (CIN4) (n=10) and invasive carcinoma (n=10). The molecular technique used was dot blot molecular assay. A positive result was generated by catalytic oxidation of peroxidase enzyme on 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate. The gold nanoparticles were used to enhance the signal produced by peroxidase activity of horseradish peroxidase (HRP) enzyme giving a more sensitive assay as compared to use of non-conjugated antibody. This study provides a significantly high and reliable diagnostic accuracy for precancerous and cancerous lesions with a sensitivity of 90%, a specificity of 90%, a likelihood ratio for positive and negative tests as 9:1 and 1:9 respectively, a Positive Predictive Value of 97.3% and a Negative Predictive Value of 69.2%. This study avails a sensitive, rapid test using paper-based nanotechnology which can be utilised in community-based screening outreaches particularly in low- and middle-income countries.

Gold nanoparticle (AuNP) bioconjugates are increasingly being utilised in biomedicine due to their low toxicity on biological tissues and unique electronic and chemical properties. They have been utilised in several biological applications namely manufacture of nanomaterials, biosensing, electron microscopy and drug delivery systems. Particularly, immuno-assays often employ gold nanoparticles (AuNPs) to enhance detection of a biological component. This paper presents a study on the bioconjugation of AuNPs with Horse Radish Peroxidase conjugated Human Papilloma Virus 16/18 Early 6 antibodies (CIP5) against Early 6 (E6) oncoprotein that is overexpressed in cervical carcinoma progression through physical adsorption. This bioconjugate can be employed in diagnostic immunoassay for cervical cancer screening. The study also demonstrated that the antibody pI, gold colloidal solution pH and amount of antibody determine the generation of stable Antibody–AuNPs bioconjugates.