Recent trends in molecular diagnostics of yeast infections : from PCR to NGS

The incidence of opportunistic yeast infections in humans has been increasing over recent years. These infections are difficult to treat and diagnose, in part due to the large number and broad diversity of species that can underlie the infection. In addition, resistance to one or several antifungal drugs in infecting strains is increasingly being reported, severely limiting therapeutic options and showcasing the need for rapid detection of the infecting agent and its drug susceptibility profile. Current methods for species and resistance identification lack satisfactory sensitivity and specificity, and often require prior culturing of the infecting agent, which delays diagnosis. Recently developed high-throughput technologies such as next generation sequencing or proteomics are opening completely new avenues for more sensitive, accurate and fast diagnosis of yeast pathogens. These approaches are the focus of intensive research, but translation into the clinics requires overcoming important challenges. In this review, we provide an overview of existing and recently emerged approaches that can be used in the identification of yeast pathogens and their drug resistance profiles. Throughout the text we highlight the advantages and disadvantages of each methodology and discuss the most promising developments in their path from bench to bedside

Ochratoxin A: General Overview and Actual Molecular Status

Ochratoxin A (OTA) is a mycotoxin produced by several species of Aspergillus and Penicillium fungi that structurally consists of a para-chlorophenolic group containing a dihydroisocoumarin moiety that is amide-linked to L-phenylalanine. OTA is detected worldwide in various food and feed sources. Studies show that this molecule can have several toxicological effects such as nephrotoxic, hepatotoxic, neurotoxic, teratogenic and immunotoxic. A role in the etiology of Balkan endemic nephropathy and its association to urinary tract tumors has been also proved. In this review, we will explore the general aspect of OTA: physico-chemical properties, toxicological profile, OTA producing fungi, contaminated food, regulation, legislation and analytical methods. Due to lack of sufficient information related to the molecular background, this paper will discuss in detail the recent advances in molecular biology of OTA biosynthesis, based on information and on new data about identification and characterization of ochratoxin biosynthetic genes in both Penicillium and Aspergillus species. This review will also cover the development of the molecular methods for the detection and quantification of OTA producing fungi in various foodstuffs

Microbial and non-microbial volatile fingerprints : potential clinical applications of electronic nose for early diagnoses and detection of diseases

This is the first study to explore the potential applications of using qualitative volatile fingerprints (electronic nose) for early detection and diagnosis of diseases such as dermatophytosis, ventilator associated pneumonia and upper gastrointestinal cancer. The investigations included in vitro analysis of various dermatophyte species and strains, antifungal screening, bacterial cultures and associated clinical specimens and oesophageal cell lines. Mass spectrometric analyses were attempted to identify possible markers. The studies that involved e-nose comparisons indicated that the conducting polymer system was unable to differentiate between any of the treatments over the experimental period (120 hours). Metal oxide-based sensor arrays were better suited and differentiated between four dermatophyte species within 96 hours of growth using principal component analysis and cluster analysis (Euclidean distance and Ward’s linkage) based on their volatile profile patterns. Studies on the sensitivity of detection showed that for Trichophyton mentagrophytes and T. rubrum it was possible to differentiate between log3, log5 and log7 inoculum levels within 96 hours. The probabilistic neural network model had a high prediction accuracy of 88 to 96% depending on the number of sensors used. Temporal volatile production patterns studied at a species level for a Microsporum species, two Trichophyton species and at a strain level for the two Trichophyton species; showed possible discrimination between the species from controls after 120 hours. The predictive neural network model misclassified only one sample. Data analysis also indicated probable differentiation between the strains of T. rubrum while strains of T. mentagrophytes clustered together showing good similarity between them. Antifungal treatments with itraconazole on T. mentagrophytes and T. rubrum showed that the e-nose could differentiate between untreated fungal species from the treated fungal species at both temperatures (25 and 30°C). However, the different antifungal concentrations of 50% fungal inhibition and 2 ppm could not be separated from each other or the controls based on their volatiles. Headspace analysis of bacterial cultures in vitro indicated that the e-nose could differentiate between the microbial species and controls in 83% of samples (n=98) based on a four group model (gram-positive, gram-negative, fungi and no growth). Volatile fingerprint analysis of the bronchoalveolar lavage fluid accurately separated growth and no growth in 81% of samples (n=52); however only 63% classification accuracy was achieved with a four group model. 12/31 samples were classified as infected by the e-nose but had no microbiological growth, further analysis suggested that the traditional clinical pulmonary infection score (CPIS) system correlated with the e-nose prediction of infection in 68% of samples (n=31). No clear distinction was observed between various human cell lines (oesophageal and colorectal) based on volatile fingerprints within one to four hours of incubation, although they were clearly separate from the blank media. However, after 24 hours one of the cell lines could be clearly differentiated from the others and the controls. The different gastrointestinal pathologies (forming the clinical samples) did not show any specific pattern and thus could not be distinguished. Mass spectrometric analysis did not detect distinct markers within the fungal and cell line samples, but potential identifiers in the fungal species such as 3-Octanone, 1-Octen-3-ol and methoxybenzene including high concentration of ammonia, the latter mostly in T. mentagrophytes, followed by T. rubrum and Microsporum canis, were found. These detailed studies suggest that the approach of qualitative volatile fingerprinting shows promise for use in clinical settings, enabling rapid detection/diagnoses of diseases thus eventually reducing the time to treatment significantly.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Innovative Biosensing Approaches for Swift Identification of Candida Species, Intrusive Pathogenic Organisms

Candida is the largest genus of medically significant fungi. Although most of its members are commensals, residing harmlessly in human bodies, some are opportunistic and dangerously invasive. These have the ability to cause severe nosocomial candidiasis and candidemia that affect the viscera and bloodstream. A prompt diagnosis will lead to a successful treatment modality. The smart solution of biosensing technologies for rapid and precise detection of Candida species has made remarkable progress. The development of point-of-care (POC) biosensor devices involves sensor precision down to pico-/femtogram level, cost-effectiveness, portability, rapidity, and user-friendliness. However, futuristic diagnostics will depend on exploiting technologies such as multiplexing for high-throughput screening, CRISPR, artificial intelligence (AI), neural networks, the Internet of Things (IoT), and cloud computing of medical databases. This review gives an insight into different biosensor technologies designed for the detection of medically significant Candida species, especially Candida albicans and C. auris, and their applications in the medical setting

Florida marine biotechnology: research, development and training capabilities to advance science and commerce

The level of activity and interest in “marine biotechnology” among Florida university faculty and allied laboratory scientists is reported in this document. The information will be used to (1) promote networking and collaboration in research and education, (2) inform industry of possible academic partners, (3) identify contacts interested in potential new sources of funding, and (4) assist development of funding for a statewide marine biotechnology research, training and development program. This document is the first of its kind. Institutions of higher learning were given the opportunity to contribute both an overview of campus capabilities and individual faculty Expressions of Scientific Interest. They are listed in the table of contents. (104pp.

Determining the Population Structure and Avirulence Gene Repertoire of the Rice Blast Fungus Magnaporthe oryzae in Kenya by Comparative Genome Analysis

Rice blast disease is caused by the ascomycete fungus Magnaporthe oryzae and is of economic importance worldwide, due to its wide geographical distribution and the severe yield losses it causes on cultivated rice. Understanding the population structure of M. oryzae is key to sustainable management of blast disease. In this study, a total of 290 M. oryzae isolates were collected from rice growing regions in Kenya including Central Kenya (Mwea irrigation scheme), Western Kenya (Ahero and Maugo irrigation schemes in Ahero and Homa-Bay respectively) and Coastal Kenya (Kwale). Initially, I undertook genotyping of a subset of Kenyan isolates by DNA sequence analysis of the internal transcribed spacer regions (ITS 1 and ITS 2) of the rRNA-encoding gene unit and by DNA fingerprinting using the Pot2 repetitive DNA element. Phylogenetic analyses based on ITS sequences clustered together isolates from Western and Coastal Kenya which were distinct from Central Kenya isolates. Cluster analysis based on 80% DNA fingerprint similarity, identified five clonal lineages designated KL1, KL2, KL3, KL4 and KL5 with most isolates belonging to lineages KL2, KL3, KL4. The clustering of isolates was region specific with Western and Coastal isolates closely related to each other and distinct from Central Kenya isolates. Distribution of mating type gene loci (MAT1.1 and MAT1.2) was determined using mating type gene specific primers. My results indicate that MAT1.1 is the predominant mating type and is distributed in all the rice growing regions of Kenya. MAT1.2 isolates were identified only in Coastal Kenya. I further undertook high throughput next-generation DNA sequencing of the genomes of 27 M. oryzae isolates from sub-Saharan Africa (SSA), including Kenya, Uganda, Tanzania, Benin, Togo, Nigeria and Burkina Faso and compared them to other sequenced strains from China, India, USA, Philippines, Thailand, Korea, Japan, France and French Guiana. Single nucleotide polymorphisms (SNPs) indicated that majority of East African isolates of M. oryzae clustered separately from West African isolates. African isolates clustered with isolates from India and China, indicating that rice blast in SSA may have originated from Asia. Pathotype analysis of Kenyan isolates was undertaken using a set of monogenic differential rice varieties, collectively harbouring 24 disease resistance genes. Rice blast resistance gene Pi-z5 conferred resistance to all the isolates tested. Other resistance genes that conferred resistance to majority of isolates tested include Pi-9, Pi-12(t), Pi-ta, Pi-ta2 and Pi-z. These resistance genes are suitable candidates for introgressing into commercially grown varieties in Kenya in combinations. I also investigated the population of M. oryzae isolates to identify cognate avirulence gene loci, including novel genes not yet reported. Finally, I evaluated rice varieties grown in Kenya for resistance to indigenous rice blast isolates under laboratory conditions. Rice variety Basmati 370 was susceptible to rice blast with varieties IR2793-80-1, BW 196, NERICA 1, NERICA 4, NERICA 10, and NERICA 11 showing some disease resistance. Varieties ITA 310 and Duorado Precoce were moderately tolerant to rice blast. This information is being used to develop a durable blast resistance strategy in sub-Saharan Africa.Halpin rice blast research scholarshipBiotechnology and Biological Sciences Research Council (BBSRC)Bill and Melinda Gates FoundationDepartment for International Development (DFID

Identification and quantification of antibiofilm metabolite extracts using electrochemical techniques

Currently 2.29% of deaths worldwide are caused by antimicrobial resistance (AMR), this is compared to 1.16% caused by malaria, and 1.55% caused by human immunodeficiency virus and acquired immunodeficiency syndrome (HIV/AIDs). Furthermore, deaths from AMR are projected to increase to more than 10 million per annum by 2050. Bacteria within biofilms have shown resistance to 1000-fold higher concentrations of antibiotics than planktonic cells. This is due to the bacteria entering a dormant-like state, reducing their growth rate. As many antibiotics target mechanisms of active metabolism, these are less effective. New antibiofilm-metabolites are needed to inhibit biofilm formation and target established biofilms. Bacteria from the marine environment are a rich, untapped source of novel bioactive metabolites, many of which have not been tested for antibiofilm properties. However, the current methods of screening for antibiofilm activity and quantification of biofilms are slow, and do not provide crucial information, such as time to eradication. This thesis aims to tap into this rich marine biodiversity. To fulfil this, strains were isolated from Scottish marine sediments, and screened these for their antibiotic and antibiofilm potential. Their metabolites were subsequently extracted and analysed using tandem mass spectrometry to identify the bioactive compound. Alongside this, we aimed to develop a method for biofilm quantification which could be translated into the clinical setting, as well as used in the screening of antibiofilm agents. This was carried out alongside crystal violet staining, as a published point of reference. The developed electrochemical techniques, electrochemical impedance spectroscopy and square wave voltammetry, were able to detect P. aeruginosa biofilm formation within an hour after seeding P. aeruginosa on the sensor. This showed that there was a 40% decrease in impedance modulus when P. aeruginosa biofilm had formed, compared to the media only control. This was also compared to a non-biofilm forming mutant, which showed only a 9% decrease in impedance modulus also compared to the media only control. As such, this thesis offers a starting point for the development of real-time biofilm sensing technologies, which can be translated into implantable materials.Currently 2.29% of deaths worldwide are caused by antimicrobial resistance (AMR), this is compared to 1.16% caused by malaria, and 1.55% caused by human immunodeficiency virus and acquired immunodeficiency syndrome (HIV/AIDs). Furthermore, deaths from AMR are projected to increase to more than 10 million per annum by 2050. Bacteria within biofilms have shown resistance to 1000-fold higher concentrations of antibiotics than planktonic cells. This is due to the bacteria entering a dormant-like state, reducing their growth rate. As many antibiotics target mechanisms of active metabolism, these are less effective. New antibiofilm-metabolites are needed to inhibit biofilm formation and target established biofilms. Bacteria from the marine environment are a rich, untapped source of novel bioactive metabolites, many of which have not been tested for antibiofilm properties. However, the current methods of screening for antibiofilm activity and quantification of biofilms are slow, and do not provide crucial information, such as time to eradication. This thesis aims to tap into this rich marine biodiversity. To fulfil this, strains were isolated from Scottish marine sediments, and screened these for their antibiotic and antibiofilm potential. Their metabolites were subsequently extracted and analysed using tandem mass spectrometry to identify the bioactive compound. Alongside this, we aimed to develop a method for biofilm quantification which could be translated into the clinical setting, as well as used in the screening of antibiofilm agents. This was carried out alongside crystal violet staining, as a published point of reference. The developed electrochemical techniques, electrochemical impedance spectroscopy and square wave voltammetry, were able to detect P. aeruginosa biofilm formation within an hour after seeding P. aeruginosa on the sensor. This showed that there was a 40% decrease in impedance modulus when P. aeruginosa biofilm had formed, compared to the media only control. This was also compared to a non-biofilm forming mutant, which showed only a 9% decrease in impedance modulus also compared to the media only control. As such, this thesis offers a starting point for the development of real-time biofilm sensing technologies, which can be translated into implantable materials

Assessment of Aflatoxin Exposure among Indian Population

What impact does dietary exposure to aflatoxins has on hepatocellular carcinoma in India? Assessing the connection between aflatoxin contamination in the diets, Hepatitis B virus infection and liver cirrhosis through Hospital based study in Andhra Pradesh, India Chronic exposure to aflatoxins was shown to cause impaired growth in children, immune suppression and liver cirrhosis, interference in micronutrient metabolism and reduced human and animal productivity. Acute severe intoxication can result in death. Most importantly, aflatoxin was shown to interact synergistically with Hepatitis B virus (HBV) / Hepatitis C virus infection. It increases the risk of hepatocellular carcinoma (HCC), one of the most common cancers in the developing countries with >400,000 new cases per annum. A joint FAO and WHO committee concluded that reduced intake of aflatoxins in HBV endemic places will have a greater impact on reducing the incidence of HCC. This requires knowledge on the level of aflatoxin exposure in populations, dietary sources of contamination and household dietary practices leading to frequent food contamination in order to identify high risk groups and to develop preventive interventions to mitigate human exposure to aflatoxins. Aflatoxins in human body are metabolized by the liver enzymes and produces aflatoxin 8,9-epoxide, which reacts with albumin in the liver to form AFB1-albumin adducts that are major biomarkers found in peripheral blood. In order to protect human health it is important to be able to estimate aflatoxin-albumin adduct in human blood. Among many methods available for estimation immunochemical methods are simple, cost effective and adaptable to situations in developing countries. Hence this study is proposed to assess the aflatoxin exposure in Indian populations using ELISA which will lead to the development of preventive interventions to minimize the risk of liver disorders and HCC. This thesis is divided into 9 chapters containing subsections. Chapter 1 and chapter 2 contains the introduction and review of literature. Chapter 3 contains materials and methods for the development of antibodies to detect aflatoxin-albumin adduct in human blood samples. Chapter 4 contains Results. Chapter 5 and 6 contains discussion, summary and conclusion. Chapter 7 contains references, Chapter 8 contains appendix and Chapter 9 contains Annexure I, II and Master charts