The application of loop mediated isothermal amplification for the detection of the sexually transmitted pathogens Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, and Trichomonas vaginalis, at the point of care.

The purpose of this multi-partnered project was the production of a fully integrated POC system, combining automated nucleic acid extraction in a centrifugally operated microfluidic disk (the LabDisk), with loop mediated isothermal amplification (LAMP) and optical detection, capable of detecting the sexually transmitted pathogens Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium and Trichomonas vaginalis in clinical urine and swab samples. LAMP is a novel nucleic acid amplification method, designed to amplify target nucleic acid in a highly specific and rapid manner, under isothermal conditions. The work detailed in this thesis presents the development of a rapid total nucleic acid extraction process, based on the capture of target nucleic acid by magnetic silica beads, optimised for use on the LabDisk platform. The extraction process was capable of the purification of target nucleic acid from a clinical sample within 5 minutes, and was robust when challenged with a range of inhibitory compounds potentially encountered in samples for STI testing. The system was capable of tolerating N. gonorrhoeae (1 x 105 CFU/ml) urine suspensions containing samples containing 50% total blood volume, 1x108 E. coli cells per ml, and 10mg/ml of BSA, without any effects on the downstream amplification time of the N. gonorrhoeae specific LAMP assay. A freeze dried lysis buffer pellet was developed, that was able to increase the sample volume, thereby decreasing the time to detection, whilst minimising the stored fluid volume on the LabDisk. LAMP assays were designed for the detection N. gonorrhoeae and M. genitalium, and the limits of detection and specificity of the assays were evaluated. The N. gonorrhoeae ORF1 assay was able to detect a minimum of 20 copies of the N. gonorrhoeae genome per reaction, whilst the M. genitalium pdhD assay was capable of detecting 16 genome copies. The tolerance of the ORF1 LAMP assay to urea, and blood, was found to be 1.8M, and 20% reaction volume, respectively. The increased tolerance of the LAMP assay to these inhibitors in comparison to PCR demonstrates the suitability of LAMP when processing urine samples for STI’s. To our knowledge this is the first application of LAMP technology for the detection of these organisms, and the first attempt at commercialising a fully integrated molecular diagnostics system based on LAMP

Prediction of inappropriate myometrial function

Preterm birth is a major clinical problem, worldwide 15000000 babies are born prematurely each year. Inappropriate myometrial function is a major cause of preterm labour. Preterm labour is the result of multiple pathological processes involving several underlying mechanisms. In all cases, a quiescent uterus in pregnancy changes to one that can produce coordinated, forceful contractions, following an increase in uterine conductivity and contractility, and cervical remodelling to facilitate cervical dilatation. Currently there are several biochemical and clinical tests available to assist in the prediction of preterm birth. Many of these have a very high negative predictive value but their positive predictive value remains low. One in five women in the UK requires induction of labour. The outcomes of this process are again difficult to predict. Both of these areas of obstetrics would benefit from improvements in prediction of clinical outcomes. Previously, phospholipase C like 1 (PLCL1) was identified as a novel intracellular protein found to be significantly downregulated in both the myometrium with the onset of spontaneous labour using sequencing techniques (Chan et al., 2014). It acts as an IP3 chelator, uncoupling phospholipase C from myometrial contractions, maintaining myometrial quiescence and therefore regulating a common pathway to inflammatory, oxytocin or prostaglandin mediated labour. We aimed to develop a clinical test utilising PLCL1 as a quiescence or susceptibility marker to other stimuli to premature labour and to determine if this marker could determine sensitivity to prostaglandins and syntocinon during the induction of labour process. During a prospective observational cohort study, patients were recruited from a preterm prevention clinic throughout mid-pregnancy, and from the antenatal ward when attending for induction of labour at term. Cervical cytobrush samples were taken to obtain cervical epithelial cells. A novel assay was developed to quantify PLCL1 from these samples. There have been various challenges in the process, including the small and varying number of cells obtained, problems with interference from cervical mucus with protein quantification and difficulty adequately lysing our cells to release the protein. We have demonstrated the presence of PLCL1 in cervical cytobrush samples using immunocytochemistry, SDS-PAGE, and western blotting and ELISAs. We have developed a method to isolate our cervical cells from the cervical mucus, lyse these cells and quantify PLCL1 using an ELISA. Our findings demonstrate that PLCL1 is a promising novel protein which could be utilised in the prediction of preterm birth and outcomes of induction of labour. As a susceptibility marker, PLCL1 could be used in conjunction with other markers

Validation of a Deployable Proteomic Assay for the Serological Screening of Sexual Assault Samples

Protein mass spectrometry (MS) has emerged as a technique to supplant traditional serological tests for body fluid identification. It was hypothesized that proteomic techniques would surpass the sensitivity and specificity of traditional serological techniques. An automated workflow coupled with protein MS has been developed for the confirmatory identification of five biological fluids. A developmental validation was completed, assessing parameters such as reproducibility, sensitivity, ion suppression, and limit of detection. Implementation was determined through tandem sample processing by MS, traditional serological tests, and standard DNA profiling methods. The MS approach offered superior detection limits while also providing true confirmatory results, producing an unambiguous identification of body fluids to the point where the technology can be considered comparable to DNA profiling. An extensive study was conducted to evaluate the effects of personal lubricants on biomarker detection in sexual assault evidence. Lubricants have the potential to inhibit protease activity, displace hydrophobic markers during solid phase extraction, and suppress ion detection during MS analysis. Three studies were performed: (1) determination of vaginal fluid biomarker detection from vaginal swabs fortified with lubricant; (2) the effect of lubricant formula on seminal fluid and saliva biomarker detection was established; and (3) the detection of biomarkers condoms. Data was interpreted by the overall peak area response (PAR) of the target biomarker, biomarker PAR in relation to internal standard, and PAR of digestion control protein. Multi-stage workflows associated with proteomic analysis remain a major hurdle towards the adoption of the technique in caseworking laboratories. A streamlined sample-to-results workflow has been developed using peptidomic analysis, allowing for straightforward preparation versus bottom up methodologies. Low molecular weight proteins were extracted and data was acquired using an orbitrap-quadrupole HRMS. Numerous protein biomarkers have been characterized in human seminal fluid, saliva, and vaginal fluid. In conclusion, the implementation of the protein MS approach offers an advantageous relationship between a positive identification and downstream DNA testing, including the capacity to deliver confirmatory contextual information in a criminal investigation. Furthermore, lubricant type does affect the ability to accurate identify protein biomarkers. And lastly, the research presented will demonstrate the use of peptidomic analysis for the confirmatory identification of biological fluids in SA type evidence

Epidemiology and prevention of sepsis in young infants and the potential impact of maternal HIV infection on neonatal sepsis

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Doctor of Philosophy Johannesburg, South Africa 2016Introduction: Neonatal infections contribute to 25% of all neonatal deaths, which account for approximately 44% of all under-5 childhood deaths globally. Pathogens responsible for sepsis in neonates and young infants can be acquired vertically prior to or during labour, or from the environment (community or hospital). This project evaluated the burden and aetiology of sepsis in neonates and young infants (≤90 days), and explored this association to in-utero exposure to human immunodeficiency virus. The study also included a specific focus on the epidemiology of invasive Group B Streptococcal disease in young infants. Additionally, we assessed the efficacy of intrapartum chlorhexidine vaginal washes for: (i) preventing early-onset neonatal sepsis; and (ii) vertical transmission of potentially pathogenic bacteria to the newborns. Furthermore, we evaluated risk factors for poor outcomes due to neonatal sepsis. Materials and methods: (i) A bacterial surveillance system was established at Chris Hani Baragwanath Academic Hospital (CHBAH) from 2004-2008 to identify young infants with bacterial sepsis hospitalised in the neonatal and paediatric wards. Medical and microbiological records were utilised to obtain clinical and laboratory data. Maternal HIV results were obtained from antenatal testing records or admission records. (ii) A blinded, randomised, placebo-controlled trial of 0.5% chlorhexidine maternal vaginal intrapartum wipes and newborn skin wipes was conducted at CHBAH between 2004 and 2007. Consented, eligible participants were randomised during labour to receive either chlorhexidine vaginal wipes or water external genitalia wipes. Newborns received either chlorhexidine full-body wipes (intervention arm) or foot wipes (control arm). Maternal and infant participants were followed up for admissions during the first month after delivery/ birth. A subset of 5144 maternal participants had an intrapartum lower vaginal swab collected, and skin swabs were collected from their newborns to assess colonisation with potentially pathogenic bacteria (Group B streptococcus, Escherichia coli and Klebsiella pneumoniae). Results: Group B streptococcus (GBS) was the most commonly isolated bacterial pathogen, causing 35.2% of culture-confirmed sepsis in infants ≤90 days, 41.6% of early-onset disease (EOD, 0-6 days), 40.5% of late-onset neonatal disease (LOD, 7-27 days) and 18.7% of young-infant community-acquired disease (YI-CAD, 28-90 days). Staphylococcus aureus (S. aureus), Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) contribute 16.2%, 12.2% and 3.4% to sepsis in young infants. Overall, incidence (per 1000 live births) of invasive GBS disease was 2.72 (95% confidence interval [95% CI]: 2.46 to 3.01), including an incidence of 1.50 and 1.22, respectively, in infants 0-6 days and 7-90 days of age. HIV-exposed infants were at greater risk of EOD (Relative risk [RR]: 1.69; 95% CI: 1.28-2.24) and LOD (RR= 3.18; 95% CI: 2.34-4.36) than HIV-unexposed infants. GBS serotypes Ia and III caused 84.0% of invasive GBS disease in young infants. Intrapartum chlorhexidine interventional wipes was not efficacious in prevention of any of: (i) vertical transmission of pathogenic bacteria (54% vs. 55%; efficacy -0.05, 95% CI: -9.5 to 7.9) to the newborns; (ii) sepsis in first 3 days of life (3% vs. 4%; p=0.65,); (iii) sepsis in the later neonatal period (both <1%; p=0.4444); or (iv) maternal puerperal sepsis(both <1%; p=0.56). Conclusion: GBS, S. aureus, E. coli and K. pneumoniae are the most commonly isolated bacterial pathogens in neonates and infants ≤90 days old. HIV-exposed infants are at greater risk of GBS sepsis. Intrapartum chlorhexidine intervention was not efficacious in reducing vertical transmission of pathogenic bacteria, neonatal or maternal sepsis. Alternative interventions to prevent sepsis in young infants, including maternal immunisation, need to be investigated in setting such as ours where there is a high prevalence of maternal HIV infection.MT201

Q-Griffithsin interactions and utility for the prevention and treatment of Mucosal infections.

Griffithsin (GRFT) is a carbohydrate binding agent (lectin) that was originally identified in the red alga Griffithsia sp. Q-Griffithsin (Q-GRFT) is an oxidation stable analog of GRFT. GRFT has demonstrated inhibitory activity against HIV-1, Coronaviruses, Hepatitis C, influenza and Ebola viruses. The broad-spectrum activity suggests the potential utility of this lectin in a wide range of viral infections. However, the lectin’s activity in mucosal infections has not been extensively studied. Using in vitro, ex vivo and in vivo assays, we have demonstrated that Q-GRFT maintains the ability to bind glycosylated ligands following incubation in murine, macaque and human rectal fluids. Additionally, we demonstrated the first reported in vitro findings of antifungal activity by Q-GRFT. Furthermore, in murine prophylaxis and therapeutic infection models, Q-GRFT was efficacious against vaginal candidiasis. In response to the ongoing COVID-19 pandemic, we have demonstrated that in engineered human cornea and airway epithelia, repeated topical application resulted in Q-GRFT accumulation in mucosal tissues. In addition, in a human cadaver study, intranasal administration resulted in adequate drug dispersion on the nasal, nasopharyngeal and oropharyngeal cavities, with the drug detected in fluids collected from these anatomic sites. These findings support the development of a protocol for a Phase 1a first-in-human intranasal Q-GRFT administration to evaluate safety, tolerability, and pharmacokinetics of the drug product for pre-exposure prophylaxis against coronaviruses. Altogether, these data demonstrated Q-GRFT’s stability in the mucosal environment and support its incorporation in multipurpose STI prevention modalities given the novel antifungal activity. Epithelial accumulation and ability to detect Q-GRFT in nasal specimens supports the feasibility of successfully performing the Phase 1a study and future drug development as a prophylaxis against coronaviruses

Contemporary Gynecologic Practice

Gynecology is frequently changing due to extensive implementation of high technology in both, the diagnosis and management of gynecologic problems. General gynecologists, gynecologic endocrinologists, infertility specialists, gynecologic endoscopists, and gynecologic oncologists will find attractive, new information in this book

Microbiology for Allied Health Students

This open textbook is a remix of Openstax Microbiology, CC-BY 4.0, and created through an Affordable Learning Georgia Round Six Textbook Transformation Grant. The textbook has the following supplemental materials within this repository: This is a collection of instructional materials for the following open textbook and lab manual: Microbiology for Allied Health Students Lab Manual Microbiology for Allied Health Students Instructional Materials Authors\u27 Description: Microbiology for Allied Health Students is designed to cover the scope and sequence requirements for the single semester Microbiology course for non-majors and allied health students. The book presents the core concepts of microbiology with a focus on applications for careers in allied health. The pedagogical features of Microbiology for Allied Health Students make the material interesting and accessible to students while maintaining the career-application focus and scientific rigor inherent in the subject matter. The scope and sequence of Microbiology for Allied Health Students has been developed and vetted with input from numerous instructors at institutions across the U.S. It is designed to meet the needs of most microbiology courses allied health students. With these objectives in mind, the content of this textbook has been arranged in a logical progression from fundamental to more advanced concepts. The opening chapters present an overview of the discipline, with individual chapters focusing on cellular biology as well as each of the different types of microorganisms and the various means by which we can control and combat microbial growth. The focus turns to microbial pathogenicity, emphasizing how interactions between microbes and the human immune system contribute to human health and disease. The last several chapters of the text provide a survey of medical microbiology, presenting the characteristics of microbial diseases organized by body system. Accessible files with optical character recognition (OCR) and auto-tagging provided by the Center for Inclusive Design and Innovation.https://oer.galileo.usg.edu/biology-textbooks/1015/thumbnail.jp