Molecular Epidemiology On Mycoplasma Infections In Female Urogenitaltract

Aim and Significance:Up to date,Mycoplasma is a wide variety of smallest prokaryotic microorganisms that can be self-replicating and are capable of be living independently. They are widely spread in human bodies,animals and plants.It has been reported that mycoplasma were easily detected in female genitourinary track,where six kinds of mycoplasma,i.e.Ureaplasma urealyticum(Uu),Mycoplasma hominis(Mh),Mycoplasma genitalium(Mg),Mycoplasma penetrans(Mpe),Mycoplasma fermentanc(Mf),Mycoplasma pirum(Mpi)were successfully isolated,which at present are thought to be closely linked with many genitourinary diseases.For recent years,the significance of studies on Mycoplasma infections has been elevated and many relevant research results have been published.However,there is an argument regarding its epidemiology,i.e.if the mycoplasma isolated from female genitourinary track has the ability to cause diseases as it has great clinical significance for the doctors to decide whether or not to have treatment.Some results have indicated that mycoplasma is a kind of opportunistic pathogen, which inhabits human specific organs,and causes disease when body's immune-response is low or local micro-environment has changed,others have suggested that only some of the special or specific strains could cause diseases,this still needs further investigations.Since late 80's last Century,the application of molecular biotechnology has remarkably facilitated its clinical diagnosis.One of the main advancements is genetic typing with rDNA sequences.It is predictable that advancement in molecular diagnosis and typing will facilitate its investigations on polymorphism analysis.This study was performing detailed typing and distributions of mycoplasma isolated from normal human female genitourinary track by analysing mycoplasma 16S rDNAs from gone library with PCR-RFLP.Samples were collected from healthy females with regular physical examination,females at early pregnancy and female outpatients,and to look at the difference in distributions of mycoplasma between them therefore correlating it to female genitourinary diseases.Methods and Materials:190 samples were collected from normal females with regular physical examination,females at early pregnancy and outpatients.The criteria for sample collection:have sex experience,no antibiotics application with two weeks,no reproductive track washing,local medicine application and no sex experience within three days just before sample taking,menstrual women were excluded.To avoid strain loss during screening,collected secretion samples were not pre-screened with incubation or immune-detection;rather they were directly tested with PCR for verification and typing.However this one-step direct PCR detection method was not widely used in clinical research.This study was performing detailed typing and distribution analysis for mycoplasma isolated from normal human female genitourinary track by analysing mycoplasma 16S rDNAs from gene library with PCR-RFLP.Based on 16SrDNAs from Gene bank,two primers GPO-and MGSO were designed as follow:GPO-3:5'-GGGAGCAAACAGGATTAGATACCCT-3' MGSO:5'-TGCACCATCTGTCACTCTGTTAACCTC-3'。The PCR product was run on a 1%agarosc gel,and a 270bp fragment will appear if it is positive.The DNA band was gel purified,followed by restriction digestions with AluⅠand RsaⅠ,and then run the gel again to compare the digestion difference to determine their corresponding RFLP types.Each type of colonies was selected for sequencing,and sequence alignments were performed against data sequences from Genebank.All results including clinical records,Leucorrhea smear and mycoplasma detection were entered EXCEL database,and analysed with statistics software STATA 8 0,choosing X~2test,,fisher's exact probabilistic method,and et al to make statistic analysis.Results:From 190 samples,Uu positive only 39,(20.53%),Mh positive only 52 (27.37%),Both Uu and Mh positive 26(13.68%),All other types were not detected.The individual ages are between 19-64,average is 34.88.There is no statistical difference for positive rate of Uu and Mh between age groups,and between healthy group and outpatient group.Also there is no statistical difference for positive rate of Uu and Mh between patients of with or without Vaginitis or Cervicitis,and is not relevant with individual's abortion times,with or without pregnancy,and whether in the state of menopause(P＞0.05).Conclusions:1:In this study,two types of mycoplasma Uu and Mh were detected,of which the positive rates were statistically irrelevant with individual ages,with or without reproductive track infections,pregnancy,abortion,and pre or post-menopause.2:The advantage of PCR diagnosis is highly specific,sensitive,and reproductive.With PCR-RFLP method,the type of mycoplasma can be accurately determined,and can be used in clinical diagnosis conveniently,quickly and accurately. Although other types of mycoplasma except Uu and Mh were not detected in this study,it is still worth a try for those strains which are difficult to grow.3:As this is just an initial study and due to the limitations on samples collections,this study result is partially biased for a certain extent.But it can be referenced for large scale studies.It also deserves further investigations on the relationship between Uu or Mh infections and those diseases like reproductive track inflammation,infertility,etc and its effects on pregnancy and infants.