Molecular Characterization of Mycoplasma genitalium Detected Among Women with Genitourinary Infections

Abstract

Mycolasmagenitalium (M. genitalium) is a gram negative bacterium that does not has cell wall. It is classified as one of sexual transmitted diseases and its infection may lead to sterility, if it is not treated in early stages. With regards to the diagnosis of M. genitalium, the nucleic acid amplification test (NAAT), polymerase chain reaction (PCR) technique is recommended to be used rather than traditional methods for instance cultural and serological techniques, as they are not accurate and precise in investigating the M. genitalium. Thus molecular technique constitutes the theme of this thesis to detect mutations of the M. genitalium among Sudanese. Two types of specimens, vaginal swab and first void urinewere collected randomly from one hundred women with genitourinary symptoms, two hundred specimens in total. These specimens were taken from two hospitals of Khartoum area, Khartoum north teaching hospital and Alhya health centre during the period 2011-2012. Prior samples collection, interviews and questionnaires were designed to collect demographic: age, sex, family history and clinical data. The urine specimens were collected in one ml phosphate buffer saline (PBS) container, whereas high vaginal swabs were collected in five ml tris-HCL to preserve DNA from damage. DNA was then extracted from all specimens and stored at -80C until time of PCR analysis. The specimens were firstly tested by real-time PCR and secondly the positive ones were confirmed, using conventional PCR for targeting domain V 23SRNA and mgpB genes. Finally, DNA sequencing was done for both genes, V 23SRNA and mgpB. This was to detect macrolides antibiotics resistance for the former and to know genotyping of M.genitaliumfor the latter. Therefore, mutation was recognized by comparison between isolated sequences versus control strains of M.genitaliumG-37. Of studied samples, 4% of Sudanese women were detected with M.genitalium. These strains were studied to recognize their single nucleotide polymorphisms (SNPs) of mgpBgene, using MgPa-13. Accordingly, six mutations were detected, four in SDN19 and two in SDN51or SDN151, when aligned with control strain sequence of M.genitalium G-37. After that, the sequences of mgpB was submitted to GenBank under accession numbers (KF612736-38) to be as a reference for further studies in the future. In conclusion,the first void urine specimen is recommended to be used for detecting M.genitalium rather than high vaginal swab. All detected polymorphisms of M.genitalium among Sudanese were sensitive to Macrolide antibiotic. The primary screening leucocyte esterase test was not sensitive in detecting M.genitalium. Therefore, PCR method is accurate and precise to diagnose patients with M.genitalium.