Abstract
The aim of the study was to determine lower genital tract carriage rate of Mycoplasma genitalium (M. genitalium) and to compare it to the carriage rates of Mycoplasma hominis (M. hominis ) and Chlamydia trachomatis (C. trachomatis) among 102 women requesting termination of pregnancy at the Horsens Hospital in Denmark. Real-Time PCR was used for the detection of bacterial DNA, and the presence of antibodies to the three microorganisms was determined by ELISA and immunoblotting. Real-Time PCR detected M. genitalium in one swab sample (0.98%) only, while the prevalence of C. trachomatis was high (15.69%) and M. hominis colonization (18.63%) was similar to colonization observed among sexually experienced adults. There was a significant difference in prevalence of M. hominis infection in the different age groups. C. trachomatis load in the cervical samples was significantly higher among young patients. There was no correlation between the presence of genital infection with C. trachomatis and genital mycoplasmas and no correlation between the presence of antibodies to these bacteria. In conclusion, in Danish patients it is not necessary to test for M. genitalium before abortion since less than 1% were found positive. The prevalence of genital C. trachomatis infections was high among the abortion-seeking patients.
| Abbreviations | ||
| M. genitalium | = | Mycoplasma genitalium |
| M. hominis | = | Mycoplasma hominis |
| C. trachomatis | = | Chlamydia trachomatis |
| PID | = | pelvic inflammatory disease |
| STI | = | sexually transmitted infections |
Introduction
In women, genital mycoplasmas are associated with various urogenital diseases. The recent in vitro organ culture study [Baczynska et al. [Citation2007]] of human fallopian tubes infected with various genital bacteria provided data that M. genitalium may cause moderate damage to the delicate human epithelium upon direct contact. Thus, estimating the prevalence of genital infection with M. genitalium is necessary especially among woman who may be at risk of genital infections. M. genitalium was found able to ascend to the fallopian tubes in vivo, and its DNA was detected in fallopian tube and endometrium specimens of a patient suffering from mild salpingitis [Cohen et al. [Citation2005]]. Presence of M. genitalium in the fallopian tubes was, however, very rare as only one specimen of 123 subjects was positive [Cohen et al. [Citation2005]].
Several studies were undertaken to establish the association of M. genitalium and upper genital tract infections resulting in endometritis and pelvic inflammatory disease (PID). Simms et al. [[Citation2003]] confirmed by PCR that 11.4% of PID-diagnosed patients were infected with M. genitalium but C. trachomatis and M. hominis were not assessed. None of the healthy controls in that study were carrying M. genitalium. The study by Uno et al. [[Citation1997]] analysed women suffering from urethritis and adnexitis and found 7.8% and 5.7%, respectively, of the patients were M. genitalium positive. In contrast, 80 pregnant asymptomatic women, also tested in that study, were all negative for M. genitalium DNA. A recent study reported a population-based prevalence estimate of M. genitalium alongside with other sexually transmitted infections (STI) in young USA adults [Manhart et al. [Citation2007]]. This study included 1,714 women and 1,218 men. More patients carried M. genitalium (1%) than N. gonorrhoeae (0.4%) but the overall prevalence of M. genitalium was low, 0.8% in women and 1.1% in men. The prevalence of C. trachomatis in this study population was 4.2%.
The aim of this study was to investigate the prevalence of M. genitalium, C. trachomatis and M. hominis in cervical swabs of pregnant Danish women seeking abortion. These women can be regarded as a higher risk population since they had unprotected sex, resulting in pregnancy and the possibility of exposure to sexually transmitted bacteria. In addition, we decided to perform this study because of the results from the previous studies examining the presence of antibodies to genital mycoplasmas and the correlation with tubal factor infertility [Clausen et al. [Citation2001]; Baczynska et al. [Citation2005]]. The infertile patients examined in the studies must have had an infection at an earlier stage in their life. In Denmark, prior to abortion women are tested for the presence of C. trachomatis and treated if positive, whereas presence of genital mycoplasmas is not determined and there are no recommendations for treatment. Our additional aim was to compare the colonization rate with M. genitalium or M. hominis to colonization with C. trachomatis in this population. This is the first study analyzing the presence of M. genitalium among patients requesting an abortion.
Results
Prevalence of Bacterial Infection Among ‘High Risk’ Abortion-Seeking Patients
Cervical samples from 102 women were collected at the gynecological investigation prior to termination of pregnancy. Women were divided into two age groups: 18 to 24 years old (younger age group) and women of 25 years or above (older age group). The older age group comprised 69.61% of all participants.
Swab samples were analysed for the presence of M. genitalium, C. trachomatis and M. hominis DNA by Real-Time PCR. The results of the tests are presented in . The presence of any of the three bacteria was detected in 31 (30.39%) samples; the most prevalent were younger women with 41.94% compared to 25.35% among the older women. There was no statistically significant difference between the groups (OR=2.11; 95% CI=0.79–5.66; p-value=0.11) despite a trend observed while using age as a continuous variable in univariate logistic regression analysis. In this case it appeared to be a marginally significant factor (p-value=0.04).
Association of Age to Genital Presence of M. genitalium, C. trachomatis, M. hominis or any of them.
M. genitalium was detected in only one (0.98%) cervical swab and with a bacterial load of approximately 83 copies/ml of the original sample. Because of the very low DNA concentration detected by Real-Time PCR in that sample (0.5 copy/μl) we repeated the PCR a total of three times. Each time the sample was detected positive with a similar DNA concentration. This patient belonged to the older age group ().
M. hominis DNA was detected in 18.63% of the women, with a bacterial load ranging from 1.66×102 copies/ml to 3.3×105 copies/ml of the original swab sample. All PCR positive samples were reconfirmed positive by culture. The prevalence was higher in the younger (32.26%) than in the older group (12.68%), and the difference in M. hominis prevalence between the two groups was significant (OR=3.24; 95% CI=1.03–10.41; p-value=0.027). Using age as a continuous variable, confirmed that age was a significant factor in the prevalence of M. hominis (p-value=0.03). Bacterial load was not significantly different between the younger and older groups (OR=1.19; 95% CI=0.14–10.54; p-value=1) ().
Bacterial Load of M. hominis and C. trachomatis Among Positive Patients.
C. trachomatis DNA was found in 15.69% of the samples. The genomic- and plasmid DNA samples were tested using the Real-Time PCR assay. The plasmid detection assay had higher sensitivity and enabled detection of low bacterial load. This was calculated from the mean concentration of C. trachomatis DNA measured by the plasmid detection assay divided by a factor 10 for determination of the genomic equivalents. The bacterial load ranged from 19 copies/ml to 7.6×106 copies/ml in the original sample. Prevalence was higher in samples from the younger group (22.58%) compared to the older (12.68%), but unlike M. hominis, there was no significant difference between the two age groups (OR=1.99; 95% CI=0.56–6.82; p-value=0.24) (). Similarly, univariate logistic regression analysis failed to show age as a significant factor (p-value=0.15).
We observed a significant difference in the bacterial load between the two age groups () in that the majority of women in the younger group had a high C. trachomatis load in their cervix (above 104 copies/ml) compared to a low load in the older group (OR=0.062; 95% CI=0.0009–0.92; p-value=0.041).
In samples from 5 women, coexistence of M. hominis and C. trachomatis was observed (). All 5 samples had a high load of C. trachomatis and a low load of M. hominis. Correlation between the presence of infection with C. trachomatis and M. hominis was, however, not significant (p-value=0.17). The sample positive for M. genitalium DNA was negative for M. hominis and C. trachomatis.
Coexistence of Genital Infection with M. hominis (MH), C. trachomatis (CT).
Detection of Antibodies Against M. genitalium, M. hominis and C. trachomatis
Presence of IgG antibodies against M. genitalium was analysed in the serum of 100 women. Sera from the remaining two women were not collected. M. genitalium antibodies were detected in 9% of the women (). There were three times more seropositive women in the older group than in the younger (11.59% versus 3.23%). There was no statistically significant difference in seropositivity between the age groups (OR=0.26; 95% CI=0.006–2.07; p-value=0.27).
Association of Age to Presence of IgG Antibodies Against M. hominis, C. trachomatis and M. genitalium.
The IgG antibodies against M. hominis were detected in 18% of the serum samples (). The distribution of antibodies to M. hominis in the different age groups was not statistically significant, 16.13% in the younger group and 18.84% in the older group (OR=0.83; 95% CI=0.21–2.82; p-value=1).
Antibodies against C. trachomatis were detected in 10% of the serum samples (). Presence of antibodies within age groups was similar, 9.68% in the younger age group and 10.14% in the older age group (OR=0.95; 95% CI=0.15–4.55; p-value=1).
None of the serum samples had IgG antibodies to both C. trachomatis and M. hominis (). One sample contained antibodies to both genital mycoplasmas, and two others had antibodies to both C. trachomatis and M. genitalium. Thus, there was no significant association between the presence of antibodies to different genital bacteria (p-value >0.05).
Coexistence of IgG Antibodies Against M. hominis, C. trachomatis and M. genitalium.
Prevalence of Genital Infection with M. genitalium, M. hominis and C. trachomatis in Relation to Presence of Corresponding Antibodies
The woman who carried M. genitalium in her cervix did not present an IgG antibody response to this microorganism. All M. genitalium seropositive women were negative for M. genitalium DNA. Among women carrying cervical M. hominis, 31.6% presented an IgG antibody response to M. hominis (). Three women positive for M. hominis DNA had IgG antibodies to C. trachomatis.
Results of Serologic Assays in Comparison to the PCR Assay for Detection of M. hominis, C. trachomatis and M. genitalium.
Only 6.25% of the women PCR-positive for C. trachomatis had C. trachomatis-specific IgG. Four women with genital presence of C. trachomatis had antibodies against M. hominis. Summarizing, there was no statistically significant correlation between the presence of genital infection and the presence of antibodies to the microorganisms (p-values >0.05).
Discussion
One hundred and two women requesting abortion at the end of 2005 and the beginning of 2006 at Horsens Hospital in Denmark were included in the study of the prevalence of M. genitalium, C. trachomatis and M. hominis in the lower genital tract. Cervical swab samples and serum samples from these women were analysed for the presence of bacterial DNA and species-specific IgG antibodies.
Among the tested swab samples only 0.98% were positive for M. genitalium, while 18.63% were positive for M. hominis and 15.69% were positive for C. trachomatis. The low prevalence for M. genitalium in our study suggests that the cervix of healthy, fertile women is only rarely colonized with this organism. This is also in agreement with previous findings [Uno et al. [Citation1997]]. Even though the number of samples examined in the present study was relatively low, cervical swabs from the women had high prevalence of both C. trachomatis andM. hominis. Thus, a study testing a larger number of samples would most probably find the same low prevalence of asymptomatic women infected with M. genitalium. That was the case in the recent population-based study [Manhart et al. [Citation2007]], where 1,714 women were tested and the prevalence of M. genitalium was only 0.8%.
In the previous Danish study, Heisterberg et al. [[Citation1985]] examining the association of genital colonization with C. trachomatis and M. hominis and the risk of postabortal infections, found 32.6% of the women had positive cultures for M. hominis and 9% harboured C. trachomatis in the cervix. In our study, the prevalence of M. hominis was lower than what was found by Heisterberg et al. [[Citation1985]] and similar to the prevalence observed among sexually experienced adults with more than three sexual partners [McCormack et al. [Citation1972]]. A possible reason for that variation could be a difference in presence of bacterial vaginosis, which was not investigated in any of the studies but could increase the prevalence of M. hominis. In addition, we found that younger patients were significantly more frequently carrying M. hominis than older patients, which could be explained by the more promiscuous sexual habits among younger adults.
The prevalence of genital C. trachomatis among abortion-seeking women was assessed in previous Danish studies, during the years 1982, 1985, 1992, and was determined to be 8–10% [Stevenson and Radcliffe [Citation1995]]. The prevalence of C. trachomatis in the present study (16.67%) is higher and could result from the use of different diagnostic techniques or from a rise in the prevalence among abortion-seeking women over the years. Even though we did not observe a significant difference between the age groups of infected patients, we observed that younger patients had significantly higher bacterial loads than older patients. This has been reported in a previous study [Eckert et al. [Citation2000]] where the bacterial load was measured in inclusion-forming units and higher unit counts related to younger age among both male and female patients.
The low correlation between the detection of genital bacterial DNA and the presence of specific antibodies was observed for both C. trachomatis and genital mycoplasmas (). Only 31.6% of women positive for M. hominis DNA had M. hominis-specific IgG antibodies. The women positive for M. genitalium did not present an antibody response to M. genitalium. Only one C. trachomatis-PCR positive woman presented an IgG response to C. trachomatis. A recent colonization of C. trachomatis by the PCR-positive women and hence sampling prior to the generation of IgG antibodies may explain this low correlation. The presence of IgG antibodies among PCR-negative women may reflect a previous C. trachomatis infection.
Finally, our results indicate that the presence of genital mycoplasmas and C. trachomatis are independent and there was no statistically significant correlation among the three microorganisms studied. Neither did the retrospective, serological analyses of antibodies to M. hominis, M. genitalium and C. trachomatis show any statistical correlation.
Materials and Methods
Patients
At the end of year 2005 and the beginning of 2006, serum and endocervical swab specimens were obtained with written consent from 102 women referred to Horsens Hospital in Denmark for termination of pregnancy. The study was approved by the local scientific ethical committee (journal no.: VF20040232). The samples were taken during the primary visit before medical or surgical abortion was performed. Women younger than 18 years of age were excluded from the study, as parental consent would have been required. Endocervical specimens were obtained and transported to the University of Aarhus as described [Baczynska et al. [Citation2004]].
Culture of Endocervical Swab Samples, DNA Extraction and Real-Time PCR for Genital Mycoplasmas
The culture of M. hominis, DNA extraction from the cervical samples and M. hominis detection by Real-Time PCR were performed as described [Baczynska et al. [Citation2004]]. In brief, endocervical specimens were obtained using a sterile chlamydial swab and the contents transferred immediately into a tube containing 2 ml of transport medium (SP-4). After transportation to laboratory facilities, 300 μl of the sample was subjected to microcentrifugation at 20,000×g, and the pellets were washed twice with PBS to remove SP-4 medium and PCR inhibitors, after DNA extraction the final volume of the DNA samples was 50 μl (up-concentrated 6 times). For detection of M. genitalium DNA, the LightCycler assay by Svenstrup et al. [[Citation2005]] was used. Since Real-Time PCR was able to detect DNA concentration as low as 1 copy/μl of either M. hominis or M. genitalium, 166 bacteria cells (assuming that 1 bacterial cell=1 genomic DNA copy) present in 1 ml of the original swab would enable successful detection. Both PCRs were optimized to be 100% specific and sensitive for detection of M. hominis and M. genitalium [Svenstrup et al. [Citation2005]; Baczynska et al. [Citation2004]]. M. hominis PCR showed 100% agreement with the microbiological culture performed on swab samples from the study population (golden standard for M. hominis diagnostics).
Real-Time PCR Assays Detecting C. trachomatis
For the detection of C. trachomatis DNA, a Real-Time PCR assay against the 7.5-kb plasmid, pCHL1, was performed on all the clinical samples and verified with an assay against the chromosomal gene CT875. C. trachomatis has approximately 7–10 plasmids per genome equivalent, and thus we assumed that one copy of CT875 gene equals 10 copies of the plasmid. CsCl2 gradient purified C. trachomatis L2 DNA was used for preparing the standard dilution series (ranging from 105 to 0.1 copy of chromosomal DNA corresponding to approximately 106 to 1 copy of the plasmid) used for quantification of the C. trachomatis DNA in the clinical samples. Primers and probes used were selected from the plasmid sequence (accession no. NC001372) and designed to obtain a 245-bp amplicon. The sequences of the primers and probes were: forward primer 5′-GCGTGTCCTGTGACCTTC-3′ and reverse 5′-AACTTATCCTCAGAAGTTTATGCAC-3′, fluorescein (FL) probe 5′-AGCAACCGCTGTGACGGAGTACA-FL-3′ and LightCycler-Red640 (LCred640) probe 5′-LCred640-ACGCCTAGGGTGCTCAGACTCC-3′(TIB MOLBIOL, Berlin, Germany). Both primers and probes were located outside of the 377 bp deletion region observed within Swedish C. trachomatis isolates [Ripa and Nilsson [Citation2007]]. The PCR reaction mixture was composed of 0.5 μM of each primer, 0.2 μM of each probe, 3 mM of MgCl2, 2 μl of ready-to-use Fast Start DNA Master Hybridization Probes (Roche Diagnostics, Mannheim, Germany), 1 μl Uracil-DNA Glycosylase (heat-labile) (Roche Diagnostics) and 2 μl of DNA template. Water was added up to 20 μl. The PCR program was composed of activation of Hotstart Taq DNA polymerase at 95°C for 10 min followed by 45 cycles consisting of denaturation 95°C for 15 s, annealing at 58°C for 8 s, and elongation at 72°C for 6 s with a temperature transition rate of 20°/s.
For the chromosomal detection assay the primer and probe sequences were selected from the CT875 gene of C. trachomatis (accession no. NP219502) and designed to amplify a 145-bp PCR product. The primer and probe sequences applied were: forward 5′-AGCAAGGGCACTATCAGGAC-3′ and reverse primer 5′-ACGGAACCCTGCTTCTAC ATC-3′, fluorescein (FL) probe 5′-CCACGTGCTAGCGACTATGATTTGCCT-FL-3′ and LCred640 probe 5′-GAAGCCCATATCCTACTCCACCTTTGCC-3′ (TIB MOLBIOL). The PCR reaction mix applied was as described for the plasmid detection assay with exception to reverse primer (1 μM) and MgCl2 (4 mM) concentrations. The PCR program was as follows, Hotstart at 95°C for 10 min, 45 cycles at 95°C for 15 s, 53°C for 8 s and 72°C for 8 s with a transition rate of 20°/s. The LightCycler instrument (Roche Diagnostics) was used.
ELISA for Detection of Antibodies Against M. hominis
Detection of IgG antibodies against M. hominis by ELISA was performed as described [Baczynska et al. [Citation2005]]. Briefly, 50 μl of serum samples, diluted 1:50 in antibody buffer (medac, Hamburg, Germany), were added in duplicate to wells in Maxisorb ELISA trays (Nunc, Roskilde, Denmark) pre-coated overnight with a mixture of membrane proteins after Triton X-114 separation of two M. hominis isolates (132 and 4195). Fifty μl of a mixture of horseradish peroxidase labelled (dilution 1:40,000) and unlabelled (0.26 μg/ml) goat-anti-human IgG (Jackson Immunoresearch Laboratories, Inc, West Grove, USA) was used as a secondary antibody. According to our cut-off value, patients with OD450 value of 1.2 and above were considered positive for presence of antibodies against M. hominis.
Immunoblotting for Detection of Antibodies Against M. genitaliu
IgG antibodies were detected by the immunoblotting method as described [Clausen et al. [Citation2001]]. Briefly, 100 μg of the whole-cell-lysate proteins of M. genitalium G37 were boiled and separated by SDS-PAGE. The proteins were then transferred to the nitrocellulose membranes. Human sera were diluted 1:200 and a rabbit control serum (immunized with the proteins from the whole M. genitalium cell extract) was diluted 1:1,000 in antibody buffer and incubated with the membrane strips. Secondary antibodies used were: alkaline phosphatase conjugated goat anti-human IgG (H+L) (Jackson Immunoresearch Laboratories, West Grove, USA) diluted 1:1,000 or goat anti-rabbit IgG (H+L) (BioRad, Hercules, CA, USA) diluted 1:3,000. MgPa membrane protein of Mycoplasma genitalium was localized on the blot by recombinant polyclonal rabbit antibody PabMgPa [Clausen et al. [Citation2001]]. The band intensities were described as weak, intermediate or strong and only patient's sera that reacted intermediately or strongly with MgPa protein were defined as seropositive.
ELISA for Detection of Antibodies Against C. trachomatis
Presence of IgG antibodies against the chlamydial Major Outer Membrane Protein (MOMP) were detected and quantified using an ELISA kit: Chlamydia trachomatis-IgG-ELISA-plus (medac, Wedel, Germany). The test was performed in accordance with the manufacturer's instructions.
Statistical Analysis
Statistical associations and correlations were examined by two-way tables, using Fisher's exact test, and by univariate logistic regression (using age as a continuous variable) and performed with the statistical software “R” (R Development Core Team, 2005).
Acknowledgments
We are very grateful to Karin Sørensen and Tina Arnbjørn for skilled laboratory practice and recruitment of patients. We thank Lisbet Wellejus Pedersen for excellent linguistic assistance and medac for providing the C. trachomatis ELISA kits. This study was supported by: “Det Sundhedsvidenskabelige Forskningsråd i Vejle Amt” (no.: 2.16.40-18), “Forskningsrådet for Sundhed og Sygdom” grant no.: 271-05-0488, and “Aarhus Universitets Forskningsfond”.
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