Genotyping And Antimicrobial Resistance Of Mycoplasma Genitalium In Male Urethritis Patients In Nanjing STD Clinic

Chapter one:Prevalence and molecular typing of Mycoplasma genitalium in male urethritis patients in Nanjing STD clinicObjective:To understand the prevalence of Mycoplasma genitalium infection in male urethritis patients in the STD clinic in Nanjing from 2018 to 2019,and to investigate the molecular epidemiological characteristics of M.genitalium infections by molecular typing.Methods:From January 2018 to December 2019,urine samples were collected from male urethritis patients who visited STD clinic of our hospital.The presence of M.genitalium was demonstrated by polymerase chain reaction(PCR)amplifying mgpB gene.M.genitalium positive samples were genotyped with mgpB-SNP,MG309-STR and the combination of the MG309-STR and mgpB-SNP methods.Phylogenetic analysis was performed based on typing results.Results:Among 652 male urethritis patients,the prevalence of M.genitalium,Neisseria gonorrhoeae and Chlamydia trachomatis were 11.3%(74/652),50%(326/652)and 28.4%(185/652),respectively.M.genitalium was identified in 18.7%(61/326)of non-gonococcal urethritis(NGU)patients and 25.6%(50/195)of Non-chlamydial non-gonococcal urethritis(NCNGU)patients.MG309 gene was successfully amplified in 62 of 74 M.genitalium positive specimens.25 different mgpB-SNP sequence types(STs)were identified in these samples(DI=0.898).and 17 STs were newly recognized.The most common STs identified in Nanjing was NJ01(25.8%,16/62).11 different MG309-STR STs were identified(DI=0.858).The ST with 11 copy numbers of repeat unit was the dominant sequence type,amounting to 25.8%(16/62).A combination of mgpB-SNP and MG309-STR yielded the highest diversity index(DI=0.987).Conclusions:M.genitalium infection was a common cause of male urethritis in Nanjing STD clinic.The genotypes of M.genitalium strains showed high diversity.NJ01 was still the dominant mgpB-SNP ST.A combination of mgpB-SNP and MG309-STR achieved high degree of discriminatory power.Chapter two:Mycoplasma genitalium macrolides and quinolones resistance in Nanjing STD clinic,2018-2019.Objective:To detect macrolide and quinolone resistance-associated mutations in Mycoplasma genitalium positive urine samples collected from male urethritis patients in the STD clinic in Nanjing during 2018 to 2019.Methods:From January 2018 to December 2019,urine samples were collected from male urethritis patients who visited STD clinic of our hospital.Macrolide resistance-associated mutations in the 23S rRNA gene and quinolone resistance-associated mutations in parC,gyrA,and gyrB genes were detected by nested PCR and sequencing.Results:74 M.genitalium-positive urine samples were collected from male urethritis patients.70(94.6%)samples were successfully amplified for 23S rRNA,69(93.2%)for parC gene,67(90.5%)for gyrA gene,and 68(91.9%)for gyrB gene.The prevalence of mutations in the 23 S rRNA,parC,gyrA and gyrB genes were identified in 91.4%(95%CI:84.83%-97.97%),84.1%(95%CI:75.47%-92.73%),16.4%(95%CI:7.5 3%-25.27%)and 2.9%(95%CI:0-6.89%)samples,respectively.The most common mutation identified in the 23S rRNA gene was A2059G(44/64,68.8%).The most common mutation in parC,gyrA and gyrB genes was G248T(40/58,69.0%;amino acid change of S83I),G285T(4/11,36.4%;amino acid change of M95I))and G1391A(2/2,100%;amino acid change of R464S),respectively.A double mutation in parC gene(G248T and T250C)was seen in one sample.60 of 74 samples were successfully amplified for 23 S rRNA,parC and gyrA genes simultaneously.No mutation was found in 3.3%(2/60)of them.83.3%(50/60)contained mutations in both 23S rRNA and parC genes.15.0%(9/60)contained mutations in all the three genes.Conclusions:Rates of macrolide resistance-associated mutations in 23S rRNA and quinolone resistance-associated mutations in parC gene remained high in male urethritis patients in Nanjing STD clinic,suggesting that routine test of antimicrobial resistance and continued surveillance of resistance in M.genitalium is warranted.Chapter three:Outcome of refractory Mycoplasma genitalium infection with long-term minocycline therapy:a case report.Objective:The purpose of this study was to evaluate the efficacy of long-term minocycline therapy in the treatment of refractory Mycoplasma genitalium infection.Methods:A refractory M.genitalium infected male patient with non-gonococcal urethritis was recruited.The patient was given minocycline(0.1g twice daily for 28 days).Test of cure and evaluation of symptoms were performed after completion of therapy at two follow-up visits.Mutations of 23S rRNA,parC,gyrA and 16S rRNA gene were determined in the patient’s urine samples.Results:Test of cure was negative for M.genitalium by PCR in this patient at two follow-up visits,and urethral discomfort completely disappeared.Macrolide resistance-associated mutation in domain V of the 23S rRNA gene(A2059G)was detected in urine samples.And the patient also contained fluoroquinolone resistance-associated mutation of G248T in parC(amino acid change of Ser83→Ile).Single nucleotide mutation in gyrA and 16S rRNA gene was not found in our case.Conclusions:Long-term minocycline therapy successfully eradicated refractory M.genitalium infection in one case.However,further expansion of the sample and more controlled studies are needed to determine the specific course,efficacy and safety of long-term minocycline therapy in M.genitalium infection.