Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 29, Num. 1, 2011, pp. 33-36

Indian Journal of Medical Microbiology, Vol. 29, No. 1, January-March, 2011, pp. 33-36

Original Article

Simultaneous and rapid differential diagnosis of Mycoplasma genitalium and Ureaplasma urealyticum based on a polymerase chain reaction-restriction fragment length polymorphism

R Mirnejad¹, N Amirmozafari², B Kazemi³

¹ Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
² Department of Microbiology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
³ Cell and Molecular Biology Research Center, Shahid Beheshti University of Medical Science, Tehran, Iran

Correspondence Address: R Mirnejad, Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran, rmirnejadreza@yahoo.com

Date of Submission: 24-Jul-2010
Date of Acceptance: 27-Nov-2010

Code Number: mb11008

PMID: 21304192

DOI: 10.4103/0255-0857.76521

Abstract

Keywords: Mycoplasma genitalium, Ureaplasma urealyticum, Polymerase chain reaction-restriction fragment length polymorphism, detection

Introduction

Mycoplasmatales are associated with infections of the genitourinary tract, reproductive failure and neonatal morbidity and mortality. ^[1],[2],[3] Mycoplasma genitalium has also been associated with pelvic inflammatory disease (PID), endometritis, arthritis and cervicitis. ^[4],[5],[6] Ureaplasma urealyticum is the main cause of non-gonococcal, non-chlamydial urethritis, acute prostatitis and acquired arthritis in men. In pregnant and non-pregnant women, Ureaplasma can cause chorioamnionitis and pre-term delivery, abortion, pre-term birth, vaginitis and cervicitis. ^{[7],[8],[9],[10],[11]} Clinical studies have demonstrated that infants born to infected mothers become infected with these bacteria, and colonization of the respiratory tract of infants has been associated with pneumonia, respiratory distress and meningitis. ^[1],[2] .

However, only few laboratories routinely culture Mycoplasmas as the required Mycoplasma media are complex and expensive and it can take 2-5 days to culture U. urealyticum and up to 8 weeks to culture M. genitalium. Identification and differentiation is traditionally achieved by serological methods that are dependent on specific antisera to each individual species ^[12]. So-called diagnostic antisera are not readily available in most laboratories and, in addition, cross-reactions may occur with some other species. Biochemical tests only allow grouping but not identification of the genital Mycoplasma isolates. At present, polymerase chain reaction (PCR) is revolutionizing diagnosis and differentiation of organisms that are difficult to cultivate. ^{[13],[14],[15],[16]} In this study, a PCR-restriction fragment length polymorphism (RFLP) assay was used for simultaneous and rapid detection and differentiation of two genital Mycoplasmas, M. genitalium and U. urealyticum, in women suffering from various genital disorders in a single amplification reaction.

Materials and Methods

Clinical specimens

Specimens were taken from a total of 210 patients (pregnant and non-pregnant women) with clinical signs who were referred to a gynaecology clinic between December 2007 and June 2008. The patients were first examined and then endocervical swab samples were collected. Women who were younger than 19 years or older than 65 years and who had had antimicrobial therapy within 2 weeks of the sampling were excluded from this study. It is explained that all patients with positive results by the gynaecologist were treated with anti-Mycoplasmic medication.

The swabs were placed in phosphate-buffered saline and transferred immediately to the laboratory for DNA estimation.

Sample preparation for PCR amplification

Extraction of DNA was performed by a high-pure PCR template preparation kit (Roche Co. , Indianapolis, IN, USA).

Oligonucleotide primers (MyUu-R and MyUu-F), which were originally designed in our laboratory, were chosen from the published nucleotide sequences of the conserved intergenic spacer region in the 16S-23S rRNA of the Mycoplasmas and were modified in order to be able to detect M. genitalium and U. urealyticum simultaneously [Table - 1].

Each PCR reaction mixture contained 15 μl master mix 1X (Ampliqon Co., Denmark), including 1X PCR buffer, 1.5 mM MgCl₂, 1 μl template DNA (0.5 μg), 0.15 mM dNTP, 1.25 U Taq DNA polymerase, 20 pmol of each forward and reverse primers and sterile distilled water up to 50 μL. PCR were performed in a GenAmp PCR system (Corbeit, Germany) according to the following program: pre-denaturation for 5 min at 95ºC followed by 30 cycles each containing denaturation at 94ºC for 30 s, annealing at 56ºC for 30 s and extension at 72ºC for 60 s, followed by a final extension at 72ºC for 5 min. ^[15]

Sample preparation for PCR-RFLP

Amplified DNA was digested [Figure - 1] using two restriction endonucleases depending on the length of the amplicon: TaqI (Fermentas, Leon-Rot, Germany) for products ranging from 227 to 332 bp and Cac8 I (Fermentas, Leon-Rot, Germany for products ranging from 72 to 393 bp [Figure - 2] and [Figure - 3].

Of the PCR products, 7 ml was digested with 4U of each enzyme, 1.5 ml 10X buffers for 2 h at 37ºC (for Cac8I enzyme) and 2 h at 65ºC (for TaqI enzyme).

Analysis of the PCR and PCR-RFLP products

The PCR and PCR-RFLP products were electrophoresed on 3% agarose gel for 1 h at 85 Volt and 25 mA, stained by SYBERgreen and visualized under a UV transilluminator. PCR products and restriction fragment sizes [Figure - 2] and [Figure - 3] were determined by comparison with the 100 bp DNA ladder, SM#333 (Fermentans, Leon-Rot, Germany). The amplification products were further evaluated by sequencing. The nucleotide sequence of M. genitalium and U. urealyticum has been deposited in GenBank under accession number in the following order: GQ367563 and GQ375145.

Statistical analysis

Statistical analysis was conducted to determine how many samples were positive for each bacterium as well as to determine those positive for two bacterial species. Perspective analyses were performed and data with rounded-up numerical values (percentage) were documented.

Results

In this study, 210 samples were collected, as described in the "Materials and Methods" section. Of the 210 patients, ranging in age from 19 to 65 years, 94.3% of were married, 67.7% did not experience any child delivery and only 2.9% had had cesarean deliveries. The DNA specimens were extracted and analysed by PCR [Figure - 1] and the amplification products were confirmed by RFLP. The restriction enzyme selection was based on our PCR product sequencing results. Briefly, the PCR product of M. genitalium was digested by Cac8I, which yielded 72 bp and 393 bp fragments, and the PCR product of U. urealyticum was digested by TaqI, which yielded 227 bp and 332 bp fragments [Figure - 2] and [Figure - 3].

Of the 210 patients that were surveyed, 100 were PCR-positive (47.6%). M. genitalium and U. urealyticum were detected by PCR in the following order: 5.2% and 44.3%, respectively. Additionally, four specimens were PCR positive for both the organisms [Table - 2].

Discussion

Rapid laboratory detection of genital mycoplasmosis in pregnant women is very important, mainly because of the ability of the bacteria to colonize the endocervical lining and cause injury to the foetus. ^[15],[16] Epidemiologic data indicated that their presence in the genital tract has been associated with the incidence of urethritis, vaginitis, cervicitis, PID, pyelonephritis and pathology of pregnancy and newborns, and, therefore, their rapid and specific diagnosis is important. ^[1],[2] In this study, PCR-RFLP was used for rapid and simultaneous detection and differentiation of two genital Mycoplasmas; M. genitalium and U. urealyticum, in clinical samples from women with genital infections in a single amplification reaction.

In this study, the rate of Mycoplasma isolation with the PCR method was 47.6%. Additionally, four cases of mixed infections that were detectable with the PCR were considerable (1.9%). The study illustrates that U. urealyticum (44.2%) infected a high percentage of the patients, whereas a lesser degree was infected with M. genitalium (5.2%). The results of this study are very similar to those of Luki et al, who used conventional PCR to detect mycoplasmosis in pregnant women and found 56.3% U. urealyticum and 3.6% M. genitalium.^[17] The minute difference in the results of these two studies may be due to the number of samples, type of subjects (pregnant vs. non-pregnant and clinical symptoms) and the target specificities of the primers used for the amplification reactions.

The results of the present study, just like those of the Stellrecht et al. study, proved that PCR is a suitable method for genital Mycoplasma detection. ^[15] The rate of Mycoplasma and Ureaplasma detection in this study was 5.7% and 44.3%, respectively, although in the Stellrecht et al. investigation it was reported to be 3.53% and 27%, respectively ^[15] . Stellrecht et al. did not detect any M. genitalium at all, and co-infection with both Mycoplasma and Ureaplasma was reported to be 2.35%. ^[15] The difference for the results of these two studies may be due to the sample collection routes as well as the methodologies of the PCRs. The PCR used for the present study employed a single primer pair specific for both the genital Mycoplasmas whereas Stellrecht et al. used multiple primer sets in their multiplex assay. Additionally, unlike the study of Stellrecht et al., the specimens used for this study were only collected from the vaginal and endocervical epithelia. ^[15]

As the PCR products were of different molecular sizes for both strains as expected, we amplified the products using specific primers, MyUu-R and MyUu-F. The digestion patterns differed markedly between the two Mycoplasma species, while resulted in identical type and field strains of each species of M. genitalium and U. urealyticum . Therefore, targeting the 16S rRNA gene for Mycoplasma PCR detection has proven to be a specific as well as a conserved target for laboratory probing of all genital Mycoplasmas.

Similar to the Hoffman et al. and Yoshida et al. studies, and in contrast with the Stellrecht et al. study, this study is unique in that only one primer pair is used for simultaneous detection and differentiation of both the genital Mycoplasmas. ^{[15],[18],[19]} Using a PCR assay with only one primer pair reduces the costs as well as the set-up time and the complexity of the reaction mixture. ^[18],[19] Designing multiple primer pairs and their set-up in a single PCR reaction mixture is rather tedious and cannot be effectively performed in every clinical laboratory. ^[16]

Summary

The PCR-RFLP assay developed in this study using only one primer pair has proven to be a simple and rapid method for the detection and differentiation of M. genitalium and U. urealyticum. Rapid detecting and differentiating of M. genitalium and U. urealyticum are clinically very significant, particularly in the management of low-birth-weight infants in whom these bacteria are a major cause of meningitis, respiratory disease and death. We therefore recommend that this PCR-RFLP assay may be used, instead of bacterial culture or serology and conventional PCR methods, for the rapid diagnosis and differentiation of genital Mycoplasma in clinical samples.

Acknowledgment

This study was supported by grant from Iran University of medical science and partially conducted in cell-molecular biology research center of Shahid Beheshti Medical Science University.

References

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