Nosocomial transmission of Mycobacterium Tuberculosis in a regional hospital

Degree of Masters of Medical Sciences (Medical Microbiology). University of KwaZulu-Natal, Durban, 2017.Nosocomial transmission of drug resistant tuberculosis is well-documented in South Africa. The level of risk varies by patient population and effectiveness of M.tb infection control measures. The risk is higher in places where large numbers of infectious M.tb patients are being treated, particularly in the absence of other infection control measures such as respiratory protection. Globalization has led to the exchange and circulation of the various strains worldwide, leading to greater global diversity of M.tb strains. This ultimately increased the risk of individuals being infected with more than one strain at a time ie; having mixed infection Methodology In this study, we investigated the extent of transmission of drug resistant M. tuberculosis between patients hospitalized in a specialized TB hospital. The study was carried out at the FOSA TB hospital, Durban, South Africa. Genotyping was performed using IS6110-RFLP and spoligotyping. Results A total of 52 patients were recruited, from which 28 isolates were successfully cultured and genotyped. Cluster analysis of the isolated genotypes suggests nosocomial transmission of drug resistant strains amongst the in-patients. A majority of the strains found among the patients belonged to the F15/LAM4/KZN family (64%), followed by three clusters of formerly uncharacterised strains which were named Unique I, II and III. Only one of the drug resistant isolates belonged to the Beijing family of strains. Several locations and activities were identified where transmission could potentially have occurred, but this could not be done for individual patients Conclusion Implementation of rapid diagnostic testing for drug-resistant TB and redesign of healthcare facilities, to minimize congregate spaces, are critical elements that should be included in TB infection control programs; in addition to administrative, environmental and personal protective measures

Development of methods for the identification of genetic polymorphism

A unique feature of the human Major Histocompatibility Complex genes is their extensive polymorphism which is localised mainly within those regions encoding the groove of the HLA molecules. Comparison of HLA allelic sequences reveals a patchwork pattern in which an individual allele comprises a unique combination of sequence motifs, each of which is shared with other alleles, and only a few alleles have a unique sequence that is not present elsewhere in the HLA region. This feature of the HLA polymorphism has complicated the application of DNA-based methods that rely on sequence identification to type the HLA genes. To overcome this problem, two novel high resolution HLA typing methods were developed. The first utilises a set of only 40 probes, which identify the recombinational motifs present in exon 2 and 3 of the HLA class I genes, allowing a unique hybridisation pattern for each allele. The unambiguous identification of the alleles is achieved by the use of a new allelic separation technique called Complementary Strand Analysis. The second method, Reference Strand mediated Conformation Analysis (RSCA), differs from conventional sequence based typing methodologies in that the HLA type is assigned on the basis of accurate measurement of conformation dependent DNA mobility in polyacrylamide gel electrophoresis. RSCA utilises a fluorescent labelled locus specific reference DNA to selectively modify the molecular conformation of the tested DNA. The use of laser based instrumentation and computer software, in addition to internal DNA markers for correction of gel variability, allows the discrimination of HLA alleles which differ by one nucleotide in a DNA fragment nearly as large as a kilobase in length. RSCA has been successfully applied in blind studies of HLA typing demonstrating that is reproducible, able to identify new alleles, and to resolve ambiguous heterozygous combinations

Defining HLA-A locus alleles from DNA using ARMS-PCR.

The HLA system is a highly polymorphic group of genes which play a central role in the immune response. The definition of the HLA system makes an important contribution to a number of fields. In the clinical setting it is a requirement for transplantation. For anthropology, the HLA system is a useful marker for assessing population groups. Functionally, it is an important component of the T cell mediated immune response. The definition offered by the established method of serology to HLA class I tissue-typing is restrictive since serology fails to discriminate between alleles which are functionally distinct. This research represents one of the first DNA based approaches and the first by PCR-SSP, to define HLA class I specificities, with particularly reference to the HLA-A locus. This thesis charts both the development and application of a robust, simple to perform, easy to interpret yet highly powerful PCR based system in which allelic definition is demonstrated. In addition to resolution, a DNA approach offers many practical advantages over the restrictive demands of a serological typing. Further more, this thesis examines the functional aspect of polymorphism, in defining the peptide binding preferences of a group of closely related HLA specificities. By so doing, this thesis shows the relevance of a highly definitive approach to HLA typing

Prevalence and intra-type variation of human papillomavirus (HPV) infection in cervical cancers: a nationwide perspective of China.

Li Chun-bong.Thesis (M.Phil.)--Chinese University of Hong Kong, 2001.Includes bibliographical references (leaves 147-169).Abstracts in English and Chinese.Abstract --- p.iDeclaration --- p.viAcknowledgments --- p.viiTable of Contents --- p.xiList of Figures --- p.xiiList of Tables --- p.xviAbbreviations --- p.xviiChapter CHAPTER 1 --- INTRODUCTION AND LITERATURE REVIEWSChapter 1.1 --- Introduction --- p.1Chapter 1.2 --- Carcinoma of the cervix --- p.6Chapter 1.2.1 --- Squamous carcinoma --- p.6Chapter 1.2.2 --- Adenosquamous carcinoma --- p.7Chapter 1.2.3 --- Adenocarcinoma --- p.8Chapter 1.3 --- Molecular biology of Human papillomavirus --- p.9Chapter 1.3.1 --- Genome structure and organization of HPV --- p.9Chapter 1.3.2 --- Expression of papillomavirus genes --- p.11Chapter 1.3.3 --- Taxonomy of HPV --- p.20Chapter 1.4 --- Diagnostic techniques in HPV detection --- p.23Chapter 1.4.1 --- Southern blot analysis --- p.23Chapter 1.4.2 --- Dot blot analysis --- p.25Chapter 1.4.3 --- In situ hybridization --- p.26Chapter 1.4.4 --- Hybird Capture System --- p.28Chapter 1.4.5 --- Polymerase Chain Reaction --- p.30Chapter 1.5 --- Human papillomavirus in cervical carcinoma --- p.33Chapter 1.5.1 --- Prevalence --- p.33Chapter 1.5.2 --- Transmission --- p.37Chapter 1.5.3 --- Risk Factors --- p.39Chapter CHAPTER2 --- MATERIALS AND METHODSChapter 2.1 --- Materials --- p.44Chapter 2.1.1 --- Chemicals and regents --- p.44Chapter 2.1.2 --- Specimens collection --- p.48Chapter 2.2 --- Methods --- p.49Chapter 2.2.1 --- Summary of methodology --- p.50Chapter 2.2.2 --- DNA extraction from fresh and paraffin embedded tissues --- p.51Chapter 2.2.3 --- Polymerase Chain Reaction using HPV Consensus Primer MY09/11 --- p.55Chapter 2.2.3.1 --- Template for PCR --- p.55Chapter 2.2.3.2 --- PCR amplification --- p.55Chapter 2.2.3.3 --- PCR product analysis --- p.56Chapter 2.2.4 --- DNA sequencing --- p.57Chapter 2.2.4.1 --- DNA sequencing reaction for ALFexpress DNA automatic sequencing --- p.57Chapter 2.2.4.2 --- ABI comparative PCR sequencing --- p.59Chapter 2.2.4.3 --- DNA sequence analysis --- p.60Chapter 2.2.5 --- Restriction Fragment Length Polymorphism --- p.61Chapter 2.2.5.1 --- Template preparation --- p.61Chapter 2.2.5.2 --- Restriction enzyme digestion --- p.62Chapter 2.2.5.3 --- Agarose gel electrophoresis analysis --- p.62Chapter 2.2.6 --- HPV Type Specific PCR --- p.63Chapter 2.2.6.1 --- Preparation of positive control DNA --- p.63Chapter 2.2.6.2 --- Preparation of HPV 52 and HPV 58 type specific PCR --- p.63Chapter 2.2.6.3 --- PCR primer design --- p.66Chapter 2.2.6.4 --- PCR amplification --- p.68Chapter 2.2.7 --- Polymerase Chain Reaction using HPV Consensus Primer GP5+/6+ --- p.71Chapter 2.2.7.1 --- Template for PCR --- p.71Chapter 2.2.7.2 --- PCR amplification --- p.71Chapter 2.2.7.3 --- PCR product analysis --- p.72Chapter 2.2.8 --- Statistical analysis --- p.72Chapter CHAPTER3 --- RESULTSChapter 3.1 --- Histology review of tumor specimens --- p.73Chapter 3.2 --- Polymerase chain reaction of HPV consensus primer MY09/11 --- p.76Chapter 3.3 --- DNA sequencing reaction --- p.81Chapter 3.4 --- Restriction fragment length polymorphism --- p.86Chapter 3.5 --- HPV type specific polymerase chain reaction --- p.90Chapter 3.6 --- Polymerase chain reaction of HPV consensus primer GP5+/6+ --- p.109Chapter 3.7 --- "Correlations of HPV prevalence, geographical variation, histology and age of the cervical cancer patients" --- p.112Chapter CHAPTER4 --- DISCUSSIONChapter 4.1 --- Prevalence of HPV infection in cervical cancer in China --- p.118Chapter 4.2 --- DNA extraction and detection methods --- p.131Chapter 4.3 --- Intratype variation of HPV --- p.141Chapter CHAPTER5 --- CONCLUSION AND FUTURE PERSPECTIVE --- p.143REFERENCES --- p.147RELEVANT PUBLICATIONS --- p.17

Role of Lactobacillus rhamnosus in Parmigiano Reggiano cheese ripening: a genotypic and post-genomic study

Lactic Acid Bacteria (LAB) constitute an heterogeneous group of bacteria that are traditionally used to produce fermented foods. They may play different roles in cheese-making where starter LAB (SLAB) participate in the fermentation process, whereas non starter LAB (NSLAB) are implicated in the maturation of cheese. Although the role of NSLAB in ripening has not yet been clarified, different authors have suggested their importance in the cheese ageing. In Parmigiano Reggiano (PR) cheese, NSLAB, autochthonic of raw milk and arising from the environment, are the protagonists of the different biochemical processes during the production and ripening stages. Recent studies in microbiological ecology of PR cheese allowed a deeper insight of the microbial composition during the manufacturing and ripening stages of the same cheese-making process. Further, the study of samples representative of the subsequent stages of the same cheese-making by culture-independent methods led to determine the microbial succession during 24 months of PR ripening. It was demonstrated that SLAB are dominant until the 2nd month of ripening. Differently, after cheese brining, the species NSLAB, especially Lb. rhamnosus, are able to grow and increase in number, while SLAB cells undergo to autolysis. Lb. rhamnosus was shown to be the dominant species present after a lack of essential nutrients, such as sugars. Therefore, this species seems to well adapt to the absence of lactose in cheese, confirming an optimal adaptability to unfavorable growth conditions. Presumably, this characteristic was due to the ability of Lb. rhamnosus to use nitrogen fraction as an alternative energy source. So far, only few studies are available about NSLAB, and in particular about Lb. rhamnosus, in PR and the exact role of these bacteria has not been investigated in greater depth. In particular, the biotypes of the dominant species during the maturation stage of cheese processing have never been studied. Indeed, it can only be hypothesized that the technological pressure determines, at different stages, the potential development of the biotype that may have a specific biochemical role leading to certain flavors and sensorial traits of the PR cheese. For this reason, Lb. rhamnosus, the dominant species among NSLAB in PR, has been chosen as subject for the present study. This Ph.D thesis aims to better understand the role played by this species in cheese-making and ripening process, and to hypothesize the strategies used to adapt its energy metabolism to the environmental conditions met in PR cheese. To reach this issue, three different steps has been pursued. In the first step, genotypic relatedness at the intraspecies level for 66 Lb. rhamnosus strains isolated during the same cycle of PR cheesemaking, was determined. Biodiversity has been estimated by means of two DNA fingerprinting techniques, i.e., randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) and repetitive extragenic palindromic-polymerase chain reaction REP-PCR analysis. It was found an intraspecies heterogeneity in Lb. rhamnosus strains which is certainly correlated to their abilities to adapt to specific environmental and technological conditions. In this regard, the detection of biotypes that mark specific steps in cheese ripening, or of those that can develop differently throughout the ripening process, suggests that they may have also specific roles closely linked to their peculiar metabolical properties. In the second step of the research the metabolic data (fermentative profiles, synthesis of organic acids and FAA) and 2-DE analyses followed by the identification of a large number of proteins were combined to highlight Lb. rhamnosus physiological mechanisms of adaptation that are responsible for survival in cheese during ripening. Cultivation was carried out under cheese-like conditions (Cheese broth) and on pasteurized and micro-filtrated milk and MRS broth As shown by fermentative profiles analysis, the major part of the Lb. rhamnosus strains had the capacity to use numerous chemical compounds as energy sources. Growth and acidification differed depending on the culture media. Compared to growth on MRS or pasteurized and micro-filtrated milk, all strains cultivated in cheese-like medium (cheese broth, CB) showed a decrease of the synthesis of D,L-lactic acid and synthesized higher levels of acetic acid. Except for one strain, the others caused an increase of the concentration of free amino acids during cultivation on CB. A proteomic approach was applied to elucidate the (i) intra-specific diversity of Lb. rhamnosus and (ii) the metabolic mechanisms involved in growth in CB which mimicked the cheese during ripening. The proteomic maps of five strains showing different metabolic traits were comparatively determined after growth on MRS and CB. The result showed that synthesis of ca. 60 - 70% of the cytosolic proteins of Lb. rhamnosus strains was not affected by the culture media. The remaining part (ca. 30-40%) of the total proteome seemed to vary depending on strains and growth media. A total of 93 protein spots which showed an increased level of synthesis during growth in MRS (46 spots) or CB (47 spot) were identified by MALDI-time of flight mass spectrometry (MALDI-TOF-MS/MS) and nano-electrospray ionization-ion trap mass spectrometry (ESI-MS/MS). Compared to cells grown in MRS, Lb. rhamnosus strains cultivated under cheese-like conditions modified the synthesis of proteins related to protein biosynthesis, nucleotide and carbohydrate metabolisms, glycolysis pathway, proteolytic activity, cell wall and exopolysaccharide biosyntheses, cellular regulation, amino acid and citrate metabolisms, oxidation/reduction processes and stress response. It was confirmed that Lb. rhamnosus strains have the capacity to activate different metabolic pathways depending on the culture media. In the last step an innovative cDNA-AFLP protocol was developed to investigate the changes in gene expression profile during the growth of one Lb. rhamnosus strain isolated in 20 month ripened PR and therefore able to growth and survive in this hostile environment. Changes in gene expression was evaluated in CB versus a rich medium (MRS). This study represents one of the few concerning bacterial transcriptomic analysis through cDNA-AFLP approaches. This technique allowed to generate unique transcript tags from reverse-transcribed messenger RNA using restriction enzymes and selective PCR amplification. Results evidenced that Lb. rhamnosus is able to modify the expression of a large part of genes when cultivated in cheese-like conditions (CB) compared to growth under in vitro optimal conditions (MRS). However, these genes still have to be recognized thus it is not possible at the moment to correlate them with proteins found with the proteomic study. For this reason further analysis will have to be focused on gel extraction followed by the identification of expressed genes. This will allow to deepen the knowledge of the basal metabolism through the identification of constitutive genes, as well as to analyze the reactions of the cells to the dairy environment by the identification of differently expressed genes in the two cultured media. Moreover the expected results could lead to the discovery of Lb. rhamnosus genes putatively involved both in the physiological mechanisms of adaptation to cheese-like substrates, that are fundamental for survival during ripening, and in the definition of organoleptic characteristics of the PR cheese

Spread of multi drug resistant tuberculosis (MDR) including extensively drug resistant turberculosis (XDR TB), in rural KwaZulu-Natal.

Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2011.Mycobacterium tuberculosis (MTB) is an airborne pathogen that is easily transmitted from person to person. An intact immune system prevents the organism from causing disease in most individuals. In South Africa, the prevalence of human immunodeficiency virus (HIV) has reached astronomical levels and is now fuelling the tuberculosis (TB) epidemic. Drug resistant MTB strains combined with a weakened host immune system is a lethal combination. Multi-drug resistant (MDR) including extensively drug resistant (XDR) tuberculosis is on the increase, with Tugela Ferry in KwaZulu-Natal South Africa, reporting the largest cluster of XDR cases in the world. It is unknown whether a single clone of the drug resistant strain is circulating in this area or whether there are multiple strains at play. Using 2 complementary genotyping methods, we showed that the MDR strains present are the result of clonal spread associated with the F28 family, as well as de novo resistance which manifests as unique patterns. The XDR epidemic in Tugela Ferry is the result of clonal spread of a strain belonging to the F15/LAM4/KZN family

The measurement of genetic diversity in mycobacterium tuberculosis using random amplified polymorphic DNA profiling

Mycobacterium tuberculosis has caused a resurgence in pulmonary disease in both developed and developing countries in recent times, particularly amongst people infected with the human immunodeficiency virus. The disease has assumed epidemic proportions in South Africa and in the Eastern Cape Province in particular. Of further concern is the isolation of increasing numbers of multiply drug resistant strains. Knowledge of the genetic capability of this organism is essential for the successful development of novel antibiotics and vaccines in an attempt to bring the global pandemic under control. Measurement of the genetic diversity of the organism may significantly contribute to such knowledge, and is of vital importance in monitoring epidemics and in improving treatment and control of the disease. This will entail answering a number of questions related to the degree of genetic diversity amongst strains, to the difference between urban and rural strains, and between drug resistant and drug sensitive strains, and to the geographical distribution of strains. In order to establish such baseline information, RAPD profiling of a large population of isolates from the western and central regions of the Eastern Cape Province was undertaken. A smaller number of drug resistant strains from a small area of KwaZulu-Natal were also analysed, with a view to establishing the genetic difference between strains from the two provinces. Cluster analysis, analysis of molecular variance and Geographical Information Systems technology were used to analyse the RAPD profiles generated. An unexpectedly high degree of genetic diversity was detected in strains from both provinces. While no correlation was seen between genetic diversity and either urban-rural situation or geographical location, a small degree of population structure could be correlated with drug resistance in the Eastern Cape. Furthermore, a significant degree of population structure was detected between strains from the two provinces, although this was still within the parameters for conspecific populations. Future work is necessary to further characterise strains from rural areas of both provinces, as well as from the eastern region of the Eastern Cape in an attempt to pinpoint the cause of the separation of the provincial populations