Frequency of Mycoplasma hominis and Ureaplasma urealyticum in Females With Urogenital Infections and Habitual Abortion History in Ahvaz, Iran; Using Multiplex PCR

AUTHORS

Susan Maleki 1 , Hossein Motamedi 1 , * , Seyyed Mojtaba Moosavian 2 , Nahid Shahbaziyan 3

1 Department of Biology, Faculty of Science, Shahid Chamran University, Ahvaz, IR Iran

2 Department of Microbiology, Faculty of Medicine, Ahvaz Jundishapur of Medical Sciences, Ahvaz, IR Iran

3 Gynecology and Obstetrics division, Imam Khomeini Hospital, Ahvaz, IR Iran

How to Cite: Maleki S, Motamedi H, Moosavian S M, Shahbaziyan N. Frequency of Mycoplasma hominis and Ureaplasma urealyticum in Females With Urogenital Infections and Habitual Abortion History in Ahvaz, Iran; Using Multiplex PCR, Jundishapur J Microbiol. 2013 ; 6(6):e10088. doi: 10.5812/jjm.10088.

ARTICLE INFORMATION

Jundishapur Journal of Microbiology: 6 (6); e10088
Published Online: August 10, 2013
Article Type: Research Article
Received: January 2, 2013
Revised: May 23, 2013
Accepted: June 1, 2013
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Abstract

Background: Infections due to Mycoplasmatales members can cause infertility, preterm delivery and neonatal morbidity and mortality. Therefore, rapid diagnosis is of great importance to control these infections and diminish their outcomes.

Objectives: The current study aimed to develop the multiplex PCR assay to detect two genital mollicutes from a single amplification reaction and the study of their relation with habitual abortion and urogenital infection in infected females.

Materials and Methods: Urine and genital samples from symptomatic females (20-54 years old) in Imam Khomeini hospital, Ahvaz, Iran, were screened for the two common mollicutes, Mycoplasma hominis and Ureaplasmaurealyticum; using multiplex PCR. The prevalence of these mollicutes in 200 healthy females was also evaluated.

Results: The results of the current study showed that the presence of M. hominis in urine (P = 0.006) and genital samples (P = 0.01) was associated with urogenital infections. Statistical analysis based on the age, revealed that the highest prevalence of M. hominis (71.4%) and U. urealyticum (60%) were in females within 30-34 and 35-39 years old, respectively. Similarly, in genital samples, the highest incidence of M. hominis (54.5%) and U. urealyticum (53.8%) was found in females within 28-33 and 34-39 years old. Interestingly, further analyses revealed direct strong relation between the mollicutes used in this study and habitual abortion as well as urogenital infections.

Conclusions: There was a strong relation between the presence of the studied M. hominis and U. urealyticum with urogenital infection in the females under study in comparison with those of control groups. The studied mollicutes were highly associated with habitual abortion in symptomatic females. The multiplex PCR was developed for simultaneous, early, and easy detection of these potential pathogens.

Keywords

Mycoplasma hominis Ureaplasma urealyticum Female Urogenital Disease Abortion Habitual

Copyright © 2013, Ahvaz Jundishapur University of Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited.

1. Background

Unlike other prokaryotes, the class mollicutes lack the cell wall and with unusually different genome are relatively small in size. The largest group is formed by the genus mycoplasma of which more than 90 species have been described (1). Mycoplasmatales are associated with infection of the genitourinary tract, reproductive failure, and neonatal morbidity and mortality. Infection with genital mycoplasmas has been linked with infertility (2-4). Ureaplasma spp. are the main cause of non-chlamydial, non-gonococcal urethritis and acute prostatitis (2). In pregnancy, Ureaplasma can cause chorioamnionitis and preterm delivery (5).

Mycoplasma homonis has been associated with pyelonephritis, pelvic inflammatory disease and postpartum septicemia (2). Diagnosis of mycoplasmal infections is usually made by serological determination or by culture. However, serological procedures are often hampered by interspecies cross reactions, and cultivation is costly since it requires special media and expertise. It can take 2-5 days to culture Ureaplasma spp. and M. hominis (2, 6). The need for an improved detection method for M. hominis and Ureaplasmaurealyticum is evident. Polymerase chain reaction (PCR) to amplify specific short segments of nucleic acid sequences is a promising rapid diagnostic test (7, 8).

2. Objectives

The current study aimed to develop the multiplex PCR assay to detect two genital mollicutes from a single amplification reaction and the study of their relation with habitual abortion and urogenital infection in infected females.

3. Materials and Methods

3.1. Bacterial Strains

The following microorganisms were purchased from the American Type Culture Collection (ATCC) and National collection of type cultures (NCTC): U. urealyticum (NCTC 10177T) and M. hominis (ATCC 23114). These microorganisms were used as positive controls.

3.2. Clinical Specimens

All specimens were received in the clinic of Imam Khomeini hospital. Specimens included 155 cervicovaginal swabs from females with vaginal discharges or genital infections and 110 urine samples from females with urine infections. Also control urine and swab samples (100 of each) were obtained from asymptomatic females. The symptomatic subjects completed standardized questionnaires about their age, educational status, any symptoms of urogenital infection and history of abortion. The swabs were transported to the laboratory in mycoplasma transport medium (PPLO broth, Difco, USA). Approximately 20 ml of clean-catch urine was then collected in a sterile plastic container. Urine samples concentration increased 10 fold by centrifugation for 30 min at 1600 ×g prior to testing. All of the specimens were stored at-70°C until DNA extraction (2).

3.3. DNA Extraction

DNA was extracted from standard strains and clinical samples as described previously by Cadieux et al. 1999 (9). Briefly, 1 ml of the sample was centrifuged at 12000 ×g for 10 min. The pellet was washed in PBS buffer and resuspended in 30 µl of dH2O. After boiling for 10 min, an aliquot of 7 µl was used directly in PCR experiments as DNA template (9).

3.4. Multiplex PCR Assay

The sequences of primers for each bacterium are listed in Table 1. Multiplex amplification reaction was prepared in 25 µl final volume containing: 10 ρM of each RNH primer and 5 ρM of each U primer (Tuba Negin, Tehran, Iran), 1.5 U Taq DNA polymerase, 1× PCR buffer, 0.2 mM dNTPs, 3 mM MgCl 2 (Cinagene, Iran), 0.1-0.5 µg DNA template and water up to 25µl. The PCR amplification was carried out using a thermal cycler (Biorad, USA) with thermal profile as follow: initial denaturation step at 95ºC for 4 min, 35 cycles at 95ºC for 50 sec, annealing temperature at 53.7ºC for 50 sec, extension step at 72ºC for 60 sec, followed by final extension at 72ºC for 7 min. The PCR products were visualized and photographed under UV light after electrophoresis for 45 min at 100 V through 1% agarose gel containing ethidium bromide (1 µg/mL).

Table 1. Nucleotide Sequences of Primers
Analysis, organism, and primerTarget or DNA sequence (5'-3')Length, bpReference
Multiplex PCR
U. urealyticumUrease gene167(10)
U9 primerGAG ATA ATG ATT ATA TGT CAG GAT CA
U8 primerGAT CCA ACT TGG ATA GGA CGG
M. hominis16S rDNA334(11)
RNH1 primerCAA TGG CTA ATG CCG GAT ACG C
RNH2 primerGGT ACC GTC AGT CTG CAA T

3.5. Statistical Analysis

Statistical analysis was performed by SPSS statistical software package V.11.5 and Chi-Square test. Statistical significance was assumed at the P<0.05 level.

4. Results

M. hominis and U. urealyticum were detected by amplifying 334 and 167 bp amplicons, respectively; using multiplex PCR (Figure 1).

Figure 1. PCR Amplification of M. hominis and U. urealyticum From Urogenital Samples.
PCR Amplification of M. hominis and U. urealyticum From Urogenital Samples.

Lane M: Marker; Lane 2: Positive control; Lane 4-8: samples positive for M. hominis; Lane 9-11: samples negative for both of the M. hominis and U. urealyticum; Lane 12: simultaneous detection of M. hominis and U. urealyticum.

M. hominis was detected in 6.4% of urine samples collected from symptomatic but no asymptomatic women (P=0.01) and in 7.1% of genital samples from symptomatic but not from asymptomatic women (P=0.006) (Table 2).

Table 2. Frequency Distribution of M. hominis and U. urealyticum in Urogenital Samples by Multiplex PCR Method
BacteriaGenital samplesUrine samples
Case group, n = 110, No. (%)Control group, n = 100, No. (%)P ValueCase group, n = 155, No. (%)Control group, n = 100, No. (%)P Value
M. hominis7 (6.4)0 (0)0.0111 (7.1)0 (0)0.006
U. urealyticum15 (13.6)8 (7.2)0.19113 (8.4)5 (3.2)0.303
Total22 (20)8 (7.2)-24 (15.5)5 (3.2)-

In urine samples, the highest frequencies of M. hominis and U. urealyticum were observed in females within 30-34 years old (71.4%) and 35-39 years old (60%), respectively (Table 3). On the other hand, in genital samples, the highest frequencies of M. hominis and U. urealyticum was observed in females within 28-33 years old (54.5%) and 34-39 (53.8%), respectively (Table 3).

In addition, the relation of habitual abortion with the presence of the studied mollicutes in either urine or genital samples was also analyzed. Surprisingly, the incidences of both mollicutes were considerably higher in urogenital samples among symptomatic females with habitual abortion history in comparison with those with no habitual abortion history (Table 4). M. hominis and U. urealyticum were recovered in 33.3% and 66.7% of genital samples, respectively; from symptomatic females with habitual abortion history (P < 0.001). Similar results were also obtained in the case of urine samples (Table 4).

Table 3. Frequency Distribution of M. hominis and U. urealyticum in the Different Age Groups
Urine samplesGenital samples
Age group, yM. hominis, No. (%)U. urealyticum, No. (%)Age group, yM. hominis, No. (%)U. urealyticum, No. (%)
20-240 (0)1 (6.7)16-211 (9.1)1 (7.7)
25-291(14.3)2 (13.3)22-272 (18.2)3 (23.1)
30-345 (71.4)2 (13.3)28-336 (54.5)1 (7.7)
35-391 (14.3)9 (60)34-391 (9.1)7 (53.8)
40-440 (0)1 (6.7)40-450 (0)1 (7.7)
45-490 (0)0 (0)46-510 (0)0 (0)
50-540 (0)0 (0)52-570 (0)0 (0)
---58-631 (9.1)0 (0)
Total7 (100)15 (100)Total11 (100)13 (100)
Table 4. Frequency Distribution of M. hominis and U. urealyticum in Urogenital Samples in Females With History of Habitual Abortion
SamplesHabitual abortion, No. (%)M. hominisP ValueU. urealyticumP Value
Genital15 (9.7)5 (33.3)P< 0.00110 (66.7)P< 0.001
Urine10 (9.1)2 (20)P = 0.0147 (70)P < 0.001

5. Discussion

The role of mycoplasmas in the genital and extragenital systems is speculative and depends on epidemiologic data ( 6 ). Clinical studies showed that mycoplasma incidence is raised in the presence of an anaerobic primer pathogen such as Trichomonas vaginalis, Chlamydia trachomatis or Neisseria gonorrhea ( 6 ). In current study, statistical analyses revealed the correlation between the incidence of M. hominis in symptomatic as well as asymptomatic females with urine (P = 0.01) and genital (P = 0.006) infections (Table 2), while no significant relation was observed between the incidence of U. urealyticum and urine (P = 0.191) and genital (P = 0.303) infections.

Mycoplasmas can grow in the stress environment created by primary pathogen. Notably, these microorganisms colonize numerously in sexually active women, but they cannot be detected due to less sensitivity of microbiological cultivation methods unless an infection occurs (11). Based on the literature, diseases such as pelvic inflammatory disease, infertility (12), habitual abortion (13), bacterial vaginosis (14), cervicitis (15), non-gonococcal urethritis (7) and chorioamnionitis (5) have been reported to be associated with M. hominis and U. urealyticum infection. Therefore, if these microorganisms are really pathogens, their early detection would be of high value. Hence, a sensitive, specific, fast, cheap and easy applicable diagnostic method is necessary.

The current study found, M. hominis and U. urealyticum in urogenital infected samples with the incidence of 6.4 to 13.6%. However, studies in other countries showed that the incidence of M. hominis and U. urealyticum was relatively higher (6, 16). Since M. hominis and U. urealyticum have been found significantly associated with low socioeconomic background, the lower incidence in the current study is not surprising, considering the low number of sexual partners among Iranians, limitations in sexual relationships for non-married people and public awareness on using contraceptive drugs (17, 18). The highest incidence of M. hominis and U. urealyticum was observed in females with urogenital infections between 30-39 years old all of whom were sexually active.

These results support previous reports on the presence of M. hominis and U. urealyticum in sexually active adults (10, 11). Apparently, the level of colonization of genital mycoplasmas is highly affected by fluctuation of estrogen and progesterone hormones (19). In addition, within this range; more sexual activity, desired condition of urogenital tract mucosa and utilization of contraceptive pills cause higher level of colonization of mycoplasmas in comparison with those of non-sexually active adults (11, 20). In the current study, 25 out of 265 females with urogenital infections had history of habitual abortion. Statistical analyses showed the direct relation between the presence of M. hominis,U. urealyticum, and habitual abortion (P < 0.001). This result was in agreement with previous report on habitual abortion in the presence of these mollicutes (13).

The current study found strong relation between the presence of the studied M. hominis and U. urealyticum with urogenital infection in females in comparison with those of the control groups. In addition, it was shown that the studied mollicutes were highly associated with habitual abortion in symptomatic females. Eventually, the multiplex PCR in the current study was developed for simultaneous, early and easy detection of these potential pathogens.

Acknowledgements

Footnotes

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