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Study of the Vaginal and Rectal Microflora in Pregnant Women, with Emphasis on Group B Streptococci

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Study of the Vaginal and Rectal Microflora in Pregnant Women, with Emphasis on Group B Streptococci Study of the vaginal and rectal microflora in pregnant women, with emphasis on Group B streptococci Nabil Abdullah El Aila Promoter: Prof. Dr. Mario Vaneechoutte Laboratory for Bacteriology Research. Department of Clinical Chemistry, Microbiology and Immunology Co-promoter: Prof. Dr. Marleen Temmerman Department of Obstetrics & Gynaecology Dissertation submitted in fulfilment of the requirements for the degree of Doctor in Biomedical Sciences Faculty of Medicine and Health Sciences, Ghent University January 2011 Nabil El Aila is supported by a PhD grant from (BOF, Bijzonder onderzoeksfonds) of Ghent University-Belgium. Dedication This thesis is dedicated to my lovely parents, Abdullah and Hanifa El Aila who taught me the value of education and have taken great pain to see me prosper in life. I am deeply indebted to them for their continued support and unwavering faith in me. It is also dedicated to my wife Aziza and my daughters: Danya, Dimah and Lana for their constant moral support, encouragement and invaluable help at every stage of my research. Members of the jury Prof. Dr. Phillip Hay Department of Genitourinary Medicine St George’s University of London, UK Prof. Dr. Pierrette Melin Belgian Reference Laboratory for Group B Streptococci Medical Microbiology Department, University Hospital of Liege Prof. Dr. Denis Pierard Department of Microbiology Vrij Universiteit Brussel Prof. Dr. Jean Plum Department of Clinical Chemistry, Microbiology and Immunology Ghent University Dr. Kristien Roelens Department of Obstetrics & Gynaecology Ghent University and Ghent University Hospital Prof. Dr. Koenraad Smets Department of Pediatrics Ghent University and Ghent University Hospital Table of the contents Members of the jury ........................................................................................................................................................... 1 Table of the contents ......................................................................................................................................................... 2 List of abbreviations .......................................................................................................................................................... 5 List of bacterial species .................................................................................................................................................... 6 Summary ................................................................................................................................................................................ 8 Samenvatting ..................................................................................................................................................................... 11 Research objectives ........................................................................................................................................................ 14 Chapter I: General introduction ................................................................................................................................. 15 I.1. Female genital tract ............................................................................................................................................. 15 I.2. Vaginal ecosystem ................................................................................................................................................ 17 I.3. The normal vaginal microflora ....................................................................................................................... 17 I.3.1. Vaginal lactobacilli ........................................................................................................................................................... 18 1.3.2. Taxonomy of vaginal lactobacilli .............................................................................................................................. 19 I.3.3. Lactobacilli and vaginal colonization resistance ................................................................................................ 20 I.3.2.1. Acid production........................................................................................................................................................ 20 I.3.2.2. Hydrogen peroxide production ........................................................................................................................ 21 I.4. Bacterial vaginosis ............................................................................................................................................... 22 I.4.1. History and microbiology .............................................................................................................................................. 22 I.4.3. Prevalence and epidemiology ..................................................................................................................................... 24 I.4.4. Pathogenesis ....................................................................................................................................................................... 25 I.4.5. Complications of bacterial vaginosis ....................................................................................................................... 26 I.4.6. Diagnosis of bacterial vaginosis ................................................................................................................................ 27 I.4.6.1. Clinical features by Amsel criteria .................................................................................................................... 27 I.4.6.2. Commercial point-of-care tests for the diagnosis of BV ......................................................................... 27 I.4.6.2.1. pH measurements: .......................................................................................................................................... 27 I.4.6.2.2. Presence of trimethylamine (electronic sensor array or electronic nose) ............................ 28 I.4.6.2.3. Testing for sialidase activity ....................................................................................................................... 28 I.4.6.2.4. Testing for proline aminopeptidase activity ....................................................................................... 28 I.4.6.2.5. DNA probe for G. vaginalis rRNA ............................................................................................................. 29 I.4.6.3. Gram stain ................................................................................................................................................................... 29 I.4.6.4. Fresh wet-mount microscopy ............................................................................................................................. 32 I.4.6.5. Treatment .................................................................................................................................................................... 34 I.5. Culture-dependent methods to study VMF ................................................................................................. 35 I.5.1. Culture media ..................................................................................................................................................................... 35 I.5.2. Phenotypic identification methods ........................................................................................................................... 36 I.5.3. Molecular identification and typing of cultured bacteria ............................................................................... 36 I.5.3.1. PCR-independent methods .................................................................................................................................. 36 I.5.3.2. Principle of tRNA intergenic polymorphism lenghth analysis (tDNA-PCR). ................................. 38 I.5.3.3. Principle of RAPD (Random Amplification of Polymorphic DNA) ..................................................... 40 2 I.6. DNA-based culture-independent methods to study VMF ...................................................................... 40 I.6.1. PCR-independent methods ........................................................................................................................................... 40 I.6.2. PCR-dependent methods ............................................................................................................................................... 42 I.6.3. Quantitative real time PCR ........................................................................................................................................... 44 I.6.3.1. DNA binding dyes ..................................................................................................................................................... 46 I.6.3.2. Hybridization probes .............................................................................................................................................. 46 I.6.3.3. Hydrolysis probe ...................................................................................................................................................... 47 I.7. Prenatal screening of Group B streptococci ............................................................................................... 50 I.7.1. Group B streptococci ......................................................................................................................................................
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