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Rapid detection and simultaneous identification of the
Zeinab Fathizadeh1, Hatave Ghasemi Tehrani2, Mohammad Kazemi3
1Department of Microbiology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
Objectives:
Mycoplasma and Ureaplasma species threaten reproductive health and fertility worldwide. Due to the lack of sensitive, accurate, and affordable diagnostic tools, the simultaneous contributions of these agents in infertility have been overlooked. This study aims to detect and identify Mycoplasma and Ureaplasma species in the genital tracts of fertile and infertile females simultaneously.
Materials And Methods:
In a case-control study, cervicovaginal clinical samples were collected from patients referred to two teaching hospitals in Isfahan from July 2019 to February 2019. The initial screening was by using Real-time PCR and designed primer to evaluate the presence of Mycoplasma and Ureaplasma species including fertile and infertile women. The bacteria species were then detected and differentiated by using the melt curve and sequenced to confirm and identify. Finally, the standard curve was used to measure and compare the copy number of each species in each group. The isolates also were detected in clinical samples using the commercial PCR method.
Results:
The frequencies of Mycoplasma genitalium and Mycoplasma hominis were (0.0, 10.0%) in the fertile group and (4.3%, 34.3%) in the infertile group, respectively. Ureaplasma parvum and Ureaplasma urealyticum species in the fertile group (7.1%, 5.7%) and in the infertile group (32.9%, 24.3) were determined, respectively. The comparison of the results obtained from PCR and Real-time PCR showed that the recent technique has the ability to track 101-103 copy numbers.
Conclusion:
The present method allows differential diagnosis and quantification of Mycoplasma and Ureaplasma species in a short time and simultaneously.
Insights
This study developed a rapid, accurate method to detect Mycoplasma and Ureaplasma in female genital tracts. The findings reveal higher prevalence in infertile women, highlighting their role in reproductive health.
Area of Science:
- Microbiology
- Reproductive Health
- Molecular Diagnostics
Background:
- Mycoplasma and Ureaplasma species are significant threats to global reproductive health and fertility.
- Current diagnostic tools lack the sensitivity, accuracy, and affordability needed to assess their simultaneous impact on infertility.
- This oversight has hindered understanding of their combined role in reproductive health issues.
Purpose of the Study:
- To develop and validate a method for simultaneous detection and identification of Mycoplasma and Ureaplasma species.
- To investigate the prevalence of these bacteria in the genital tracts of both fertile and infertile females.
- To quantify the bacterial load of each species in relation to fertility status.
Main Methods:
- A case-control study involving cervicovaginal samples from fertile and infertile women.
- Real-time PCR with specifically designed primers for initial screening and detection.
- Melt curve analysis and sequencing for species differentiation and confirmation.
- Standard curve method for quantifying bacterial copy numbers.
- Validation using commercial PCR methods.
Main Results:
- Mycoplasma genitalium and Mycoplasma hominis were detected at higher frequencies in infertile women (4.3%, 34.3%) compared to fertile women (0.0%, 10.0%).
- Ureaplasma parvum and Ureaplasma urealyticum were also significantly more prevalent in infertile women (32.9%, 24.3%) than in fertile women (7.1%, 5.7%).
- Real-time PCR demonstrated sensitivity in tracking bacterial loads from 10^1 to 10^3 copy numbers.
Conclusions:
- The developed Real-time PCR method enables rapid, simultaneous differential diagnosis and quantification of Mycoplasma and Ureaplasma species.
- The study confirms a higher prevalence of these urogenital mycoplasmas in infertile females.
- This diagnostic approach can aid in understanding the contribution of these pathogens to infertility and reproductive health.
