Studies On Molecular Epidemiology And Macrolide Resistance Of Mycoplasma Genitalium

Mycoplasma genitalium infection is a newly established cause of nongonococcal urethritis (NGU) among heterosexual men in the past decade and is associated with mucopurulent cervicitis (MPC) and pelvic inflammatory disease (PID) in women. However, few studies have focused on the spectrum of M genitalium infection and the prevalence of Mgenitalium infection from anatomical sites other than urethra. Men who have sex with men (MSM) is one of the high risk populations for sexually transmitted diseases (STD) including M genitalium. Orogenital sex and anal sex were common practice among the population and it indicates of M genitalium infection at rectal and pharyngeal sites. Genotyping is an important tool for understanding the pathogenesis and epidemiology of M genitalium and mgpB single nucleotide polymorphism (SNP) is the most popular typing method for M genitalium. To investigate the characteristics of M genitalium infections, mgpB SNP typing method was performed to analyze the M genitalium infections from sexual partners and from different anatomical sites of the same patients. As azithromycin was widely used for the treatment of M genitalium infections, researchers have found that macrolide resistance in M genitalium can be induced by insufficient therapy with single-dose azithromycin and the resistance is strongly associated with point mutations in domain V of the23S rRNA gene. In the current study, a cross-sectional study was conducted to understand the epidemiology of Mgenitalium in Nanjing areas, especially from anatomical sites other than urethra. mgpB SNP typing method was performed for the M genitalium positive specimens from NGU/MPC patients in the STD clinic of the institute of Dermatology, Chinese Academy of Medical Sciences&Peking Union Medical College and from MSM mentioned above. Mutations in domain V of the23S rRNA gene were tested for the M genitalium positive persistent or recurrent urethritis patients who have suffered macrolide treatment failure. The current study was divided into three parts as follows:Part one:The infection of M genitalium at various anatomical sites of men who have sex with menWe aimed to determine the prevalence of M genitalium and C trachomatis in urethra, rectum and pharynx of MSM attending gay bars in Nanjing and the surrounding areas, and to analyze the association between the agents detection and clinical manifestations. A cross-sectional study of388MSM attending10gay bars in five cities in Jiangsu Province was conducted between September2008and February2009. Rectal and pharyngeal swabs and first void urine (FVU) were tested for M genitalium and C trachomatis by polymerase chain reaction. Bivariate and multivariable analyses were performed to determine the association between M genitalium and C trachomatis infections and their clinical manifestations. The results showed that the prevalence of M genitalium infection at urethral, rectal and pharyngeal sites was17.2%(95%Confidence Intervals [CI]:13.4%to21.0%),11.8%(95%CI:8.4%to15.2%), and13.5%(95%CI:9.9%to17.1%), respectively. Urethral M genitalium infection was significantly associated with urethral discomfort in the past3months (adjusted odds ratio [AOR]:2.22,95%CI:1.09-4.52) and polymorphonuclear leucocyte (PMNL) counts per high-power microscope field (AOR:2.40,95%CI:1.02-5.62). Rectal M genitalium infection was independently associated with rectal discharge in the past3months (AOR:6.06,95%CI:1.59-23.11). C trachomatis was more commonly detected in rectum (16.0%,95%CI:12.2%to19.8%) than in urethra (9.4%,95%CI:6.4%to12.3%) and in pharynx (0.8%,95%CI:0.1%to1.6%). For C trachomatis infection, PMNL counts per high-power microscope field (AOR:4.66,95%CI:1.80-12.07) and having receptive anal intercourse with a male in the past3months (AOR:2.27,95%CI:1.14-4.54) were associated with urethral and rectal C trachomatis infection, respectively. It indicated that high prevalence of M genitalium infection was observed among MSM in Nanjing and the surrounding areas at urethral, rectal and pharyngeal sites. M genitalium infection was significantly associated with urethral and rectal symptoms. C trachomatis was still one of the main causes of infection at urethral and rectal sites.Part two:Studies on genotying and molecular epidemiology of MgenitaliumOur purpose was to investigate the genotypes of M genitalium detected in MSM and NGU/MPC patients in Nanjing and the surrounding areas.149M genitalium positive specimens from MSM detected in Chapter one and71from NGU/MPC patients were genotyped by mgpB SNP method and analyzed using Bioinformatics software. The results showed that46sequence types were identified by mgpB SNP method, of which37were novel. NJ01and genotype1were the most common sequence types in Nanjing and the surrounding areas. Of21MSM who were detected M genitalium positive at two anatomical sites,10have matching genotypes at the two sites. Multiple sex partners as well as no immune protection acquired from the current status of infection may be causes of different M genitalium strains infected in different anatomical sites of the same patients. And of three concurrently M genitalium infected couples, two couples had matching genotype profiles. The findings indicated that there was high genetic diversity of M genitalium in Nanjing and the surrounding areas and the type distribution of M genitalium in these areas was different from other areas.Part three:Detection of Point Mutations Associated with Mgenitalium Resistance to MacrolideOur objective was to investigate the molecular mechanism of M genitalium resistance to macrolide. Ninty one persistent or recurrent urethritis patients who have suffered macrolide treatment failure were tested for M genitalium, C trachomatis, N gonorrhoeae and U urealyticum. Domain V of the23S rRNA gene of the M genitalium positive specimens was amplified by nested PCR followed by direct automatic sequencing. The DNA sequences were compared to the sequence of M genitalium G37to find molecular mechanisms of macrolide resistance. The results showed that21specimens were M genitalium positive, of which18specimens amplified domain V of the23S rRNA gene. Seventeen specimens harbored a point mutation respectively, among them,10had an A to G transition at position2059,5had an A to G transition at position2058, the other2specimens showed an A to T transition at position2058. No significance was found between the23S rRNA gene mutation and mgpB SNP type. The findings indicated that M genitalium infection was one of the causes of persistent or recurrent urethritis. The point mutation in23S rRNA may contribute to the macrolide resistance of Mgenitalium in Nanjing and the surrounding areas.