MicroRNAs (miRNAs) have now been identified in a vast array of organisms and a great deal of research has been carried out to elucidate the role they play. The dysregulation of miRNA expression has been implicated in a number of disease states and their importance has been highlighted by the beginning of their utilisation as therapeutics. The focus of this study was to identify the role played by miRNAs encoded by the Marek’s disease vaccine viruses, Marek’s disease virus serotype 2 (MDV-2) and Herpesvirus of turkeys (HVT). In order to better understand the functions of these miRNAs we wanted to identify their targets within the host cell. Using a combination of bioinformatic and biochemical approaches we were able to build up a library of potential targets. Three viral miRNA targets; AKT3, RAP1A and DEK, were further validated using dual-luciferase assays to highlight the exact site of miRNA targeting, and western blots to demonstrate an effect of miRNA targeting on protein abundance. An attempt at using label-free proteomics to observe the viral miRNA mediated changes in the host proteome is also described, however this proved to be unsuccessful. Additionally the function of one particular MDV-2 miRNA, mdv2-miR-M21, was explored in more detail, describing its role as a potential ortholog of the host miRNA; gga-miR-29b. By using the observation that the viral miRNA contained an identical 'seed' region to the host miRNA, we were able to use the data collected from existing studies on miR-29b to search for targets of mdv2-miR-M21. We demonstrated that mdv2-miR-M21 targeted DNMT3B, crucial for epigenetic modification of the genome. The final part of this study aimed to understand the wider context the viral miRNAs played in the viral biology and protective ability of the vaccine viruses. The miRNAs were deleted from the viruses, and then the miRNA-deletion viruses were used to vaccinate birds before challenge with the oncogenic Marek's disease virus serotype 1 (MDV-1), survival rates to the 'wild-type' MDV-2 and HVT vaccine viruses were then compared.

Introduction: Toxoplasmosis is caused by infection with the protozoan Toxoplasma gondii (T. gondii), an obligate intracellular parasite. T. gondii infects a large portion of the world’s population, but uncommonly causes clinically significant disease. Those that are at greatest risk for more severe disease with toxoplasmosis are the immunologically impaired, fetuses, and newborns. T. gondii infection in immunocompetent patients can present as a self-limiting acute infection, or as an acute systemic disease. There are three main T. gondii genotypes, I, II, and III, with varying geographical prevalence. T. gondii is most commonly acquired via ingestion of infectious oocysts, from the environment, tissue cysts from contaminated food items, vertical transmission, or via organ transplantation from an infected donor. Diagnosis can be made via histological and serologic testing in suspected patients. Seropositive testing should be considered within the clinical context, as IgM antibodies may persist for months to years. IgG antibody avidity patterns further help delineate acute versus chronic infections. Histopathology from tissue biopsy of lymphadenopathy is more commonly pursued to establish diagnosis in immunocompetent patients. Case Report: We present a 37-year-old male who presented to the clinic with persistent bilateral non-tender occipital lymphadenopathy of two months duration. Patient also endorsed an acute fluid filled blister on the penis, recurrent cold sores, and significant fatigue. Review of systems were unremarkable. Patient’s immunizations were up-to-date. Patient is an active military serviceman with history of overseas deployment. Patient reports consuming undercooked meat overseas, as well as game meat preparation while hunting. Similar symptoms were also reported by another fellow veteran. Laboratory studies revealed normal CBC, CMP, and TSH. HIV, gonorrhea, and chlamydia testing were negative. Urology referral found no abnormalities. Aspiration biopsy of the right occipital lymph node demonstrated granulomas and aggregates of histiocytes compatible with reactive hyperplasia. Findings were suggestive of toxoplasmosis and no malignancy was found. Follow up T. gondii serological testing results revealed Ab IgM: 104 AU/ML (reference range 0.0-7.9). Toxoplasma gondii Ab IgG: >400 AU/ML (reference range 0.0-7.1), which were consistent for active infection. Patient was referred to Infectious Disease and supportive therapy was recommended. A three month follow up showed improvement in symptoms. Discussion: Although acute infections with T. gondii in immunocompetent patients typically are self-limiting, more serious systemic infections may occur. A pyrimethamine-containing antibiotic regimen is recommended for treating systemic infections. We propose educating high-risk individuals with appropriate preventive measures, which may be beneficial in preventing Toxoplasmosis.

Infectious diseases from novel viruses have become a major public health concern. Rapid identification of virus–host interactions can reveal mechanistic insights into infectious diseases and shed light on potential treatments. Current computational prediction methods for novel viruses are based mainly on protein sequences. However, it is not clear to what extent other important features, such as the symptoms caused by the viruses, could contribute to a predictor. Disease phenotypes (i.e., signs and symptoms) are readily accessible from clinical diagnosis and we hypothesize that they may act as a potential proxy and an additional source of information for the underlying molecular interactions between the pathogens and hosts. We developed DeepViral, a deep learning based method that predicts protein– protein interactions (PPI) between humans and viruses. Motivated by the potential utility of infectious disease phenotypes, we first embedded human proteins and viruses in a shared space using their associated phenotypes and functions, supported by formalized background knowledge from biomedical ontologies. By jointly learning from protein sequences and phenotype features, DeepViral significantly improves over existing sequence-based methods for intra- and inter-species PPI prediction. Lastly, we propose a novel experimental setup to realistically evaluate prediction methods for novel viruses.

Doctor Pharmaceuticae - DPharm / Treatment of the human immunodeficiency virus (HIV) and tuberculosis (TB) infections have become very complicated due to the advent of drug resistance. Drug combinations offer an alternative approach to reducing the emergence of drug resistance. Pharmaceutical co-crystals have provided the pharmaceutical industry with the ability to optimise the physicochemical properties of active pharmaceutical ingredients (APIs) while preserving the biological activity. Pharmaceutical co-crystals are formed between APIs and suitable co-formers that are biologically safe or even a second or third API.

Anemia is known to worsen treatment outcomes in malaria, but there are not many studies to identify the predictors of anemia in Nigerian children with malaria. The objective of this study is to identify some of those predictors. Socio-demographic variables are predictors of anemia among under five children in Nigeria was the hypothesis tested. This is a cross-sectional study that used the 2018 demographic health survey (DHS) data from Nigeria to explore some of the factors that determine the presence of malaria-anemia co-morbidity in Nigerian children less than five years (N= 265). The outcome variable was anemia status in children under five with malaria and the explored predictors include age, sex, residential type, region of residence, mother’s education status and family’s wealth index. The study analyzed unweighted and weighted frequencies of the variables and conducted comparison of the outcome groups based on the predictor variables using Chi-square. Univariable and multivariable logistic regression was used to explore the strength of relationship between the outcome variable and the significant predictor variables in bivariate analysis. SAS 9.4 was used for the statistical analysis. Analysis of weighted frequencies showed that 55% of the children were less than 2 years of age while the sex was almost equally distributed between males and females (50.9% vs 49.1%). Just over two-thirds lived in a rural area, 63.2% resided in the Northern part of the country, 59.1% had a rich family and majority (69.1%) had anemia. When cross-tabulated with the outcome variable of anemia status, there was a significant difference in the categories of age (P=0.0048), residential type (P=0.0031), mother’s education status (P=0.0210) and family’s wealth index (P=0.0010). Univariable logistic regression showed that children less than 2 years had over two times higher odds of developing anemia when infected with malaria compared to older children aged 3-4 years (OR:2.17, 95% CI:1.26-3.74, P=0.0052). Urban-dwelling children had 57% reduced odds of developing anemia compared to rural-dwelling children (OR:0.43, 95% CI:0.25-0.76, P=0.0034). Children of educated mothers had 50% reduced odds of developing anemia compared to children of uneducated mothers (OR:0.50, 95% CI:0.28-0.91, P=0.0222), while children in poor families had 165% increased odds of developing anemia compared to those born into rich families (OR:2.65, 95% CI 1.47-4.78, P=0.0012). Once adjusted for all significant variables in the bivariate analysis, only age remained significant as a predictor of anemia in children under five years with malaria (OR:2.29, 95% CI:1.31-4.02, P=0.0039). Younger age seems to be an important predictor of anemia in Nigerian children with malaria in real life settings given its significance on the multivariable model. This finding should inform clinicians on the need to pre-empt and treat anemia in Nigeria’s younger children with malaria for better treatment outcome.

In the rural areas of Kwa Zulu Natal small scale farming is the main agricultural activity, which is often carried out in addition to other employment taken on in another location by at least one member of the household. Although Veterinary services (VS) was aware of several diseases occurring in this area and has implemented a dipping strategy for tick borne diseases as well as a regional annual vaccination campaign for Rabies, Anthrax and Black quarter, uncertainty remained about the relative importance of other diseases such as brucellosis, leptospirosis and enzootic bovine leucosis. Further it was of interest to investigate the serological resistance of cattle against the tick borne diseases babesiosis and anaplasmosis. In addition to this veterinary services wanted to increase their understanding of the perceptions and practices of local cattle owners that were relevant for the context of disease control. A serological survey of cattle was carried out between March 2001 and December 2003 to determine the prevalence of the above-mentioned diseases. The survey was designed as a two-stage survey, considering the diptank as the primary sampling unit. The conduction of the questionnaire survey was based on a convenience sample and took place during the dipping procedure. The apparent prevalence at district level was adjusted for clustering, and diagnostic test sensitivity and specificity and displayed in maps. The mean true prevalence of brucellosis varied from zero to 15.8 percent in the north eastern region with the large majority of the districts being disease free. Enzootic Bovine Leukosis (EBL) was widely present in the province at generally low prevalences, except in the central region where the highest prevalence at district level was recorded to be 70 percent. Leptospirosis also occurred frequently with the highest prevalence noted being 62 percent at district level. The southern regions showed a higher leptospirosis prevalence than other areas of the province, while in some of the northern and western districts a lower prevalence is noted. The encountered servovars were pomona, which occurred most frequently, tarrasovi, bratislava, hardjo, canicola and icterohaemorrhagica. While Babesia bovis and anaplasmosis occurred at a high prevalence throughout the province, B. bigemina was found to be much less established and is absent from many of the northern districts. Most prevalences calculated at district level do show large confidence intervals due to uncertainty that arose from the sampling frame and should be interpreted with care. / Dissertation (MSc (Production Animal Studies))--University of Pretoria, 2007. / Production Animal Studies / MSc / unrestricted

Presentation: A 3 month-old male is referred for admission with a 2-day history of fever, having been diagnosed with pneumonia and prescribed a cephalosporin on the previous day. A blood culture obtained at that time is positive for coagulase negative Staphyloccocus. On exam, he is ill-appearing. He has bilateral conjunctivitis that spares the limbus, non-exudative pharyngitis, and a polymorphic truncal rash. There is no appreciable cervical lymphadenopathy or extremity involvement. A chest x-ray demonstrates a round infiltrate of the left upper lobe, and initial labs reveal a white blood count of 17.5, a C-reactive protein (CRP) of 23.9 mg/dL, and a normal comprehensive metabolic panel. His positive blood culture is deemed a contaminant, and antibiotic coverage for community-acquired pneumonia is given with ampicillin. Diagnostic evaluation: On day 5 of illness, his fevers persist despite broadened antibiotic coverage. Further work-up has ruled out viral respiratory pathogens and Epstein-Barr virus as a cause of persistent fevers. Incomplete Kawasaki disease is suspected due to continued fevers, the presence of three clinical criteria, and further increase in his CRP. He lacks other supplemental laboratory criteria, so an echocardiogram is obtained that shows mild dilation of the left anterior descending artery (LAD) of indeterminate significance. A repeat echocardiogram 2 days later reveals progressive dilation of left main coronary artery (LMCA), LAD, and right coronary artery (RCA). Diagnosis: Dilation of the LAD and RCA confirm a diagnosis of incomplete Kawasaki disease. Within 48 hours of treatment with IVIG and high-dose aspirin, the patient is afebrile with resolving symptoms and a declining CRP. He is discharged on the 9th day of illness on low dose aspirin and a cephalosporin to complete an antibiotic course for concurrent pneumonia. Conclusion & Discussion: This case illustrates the importance of maintaining a high index of suspicion for an incomplete presentation of Kawasaki disease, particularly among infants. The American Heart Association’s guidelines were updated in 2017 to improve recognition of incomplete Kawasaki disease, particularly among infants who are more likely to have an incomplete presentation, abnormalities of the coronary arteries, and a delayed diagnosis. The key to this patient’s diagnosis was the presence of a bilateral conjunctivitis that spared the limbus. A bilateral, non-exudative conjunctivitis that spares the limbus has been recognized as a feature suggestive of Kawasaki disease for the better part of four decades; our review of the literature suggests this feature is highly specific to the diagnosis of Kawasaki disease.

Staying current on literature related to antimicrobial stewardship can be challenging given the ever-increasing number of published articles. The Southeastern Research Group Endeavor (SERGE-45) identified antimicrobial stewardship–related peer-reviewed literature that detailed an actionable intervention for 2019. The top 13 publications were selected using a modified Delphi technique. These manuscripts were reviewed to highlight the actionable intervention used by antimicrobial stewardship programs to provide key stewardship literature for teaching and training and to identify potential intervention opportunities within one’s institution.

With an increasing number of antimicrobial stewardship-related articles published each year, attempting to stay current is challenging. The Southeastern Research Group Endeavor (SERGE-45) identified antimicrobial stewardship-related peer-reviewed literature that detailed an "actionable" intervention for 2017. The top 13 publications were selected using a modified Delphi technique. These manuscripts were reviewed to highlight the "actionable" intervention used by antimicrobial stewardship programs to provide key stewardship literature for training and teaching and identify potential intervention opportunities within their institutions.

Objective: Use of ampicillin in outpatient parenteral antimicrobial therapy (OPAT) has historically been complicated by frequent dosing and limited stability. The purpose of this study was to evaluate stability of ampicillin using high-pressure liquid chromatography (HPLC) in an OPAT dosing model using continuous infusion at room temperature over 24 hours immediately following preparation compared with batches stored under refrigeration for 24 hours, 72 hours, and 7 days. Methods: An HPLC method was developed and validated as stability indicating using guidance in USP general Chapter <1225>. Four ampicillin batches were prepared for each experimental condition (immediate use and refrigerated storage for 24 hours, 72 hours, and 7 days). A pump was used to recirculate the solutions through medical-grade tubing for 24 hours. Triplicate 1-mL aliquots were removed from each batch at time 0, 4, 8, 12, and 24 hours and analyzed for ampicillin concentration. Results: Each batch was assayed for initial concentration (20.34-21.50 mg/mL), and percent recovery compared with that concentration thereafter. For the duration of infusion, the average recoveries were 96.4%, 95.8%, 94.6%, and 90.3% for immediate use, 24-hour storage, 72-hour storage, and 7-day storage, respectively. The recovery remained above 90% for all batches and time points, except for 7-day storage, which fell below 90% after 4 hours of circulation. Conclusion: Ampicillin can be prepared and stored in a refrigerator for up to 72 hours prior to continuously infusing at room temperature over 24 hours with less than a 10% loss of potency over the dosing period. This model supports twice weekly OPAT delivery of ampicillin.