JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2010, p. 3624–3627 0095-1137/10/$12.00 doi:10.1128/JCM.00232-10 Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 48, No. 10
Mycoplasma genitalium PCR: Does Freezing of Specimens Affect Sensitivity?䌤 Katrina Hutton Carlsen and Jorgen Skov Jensen* Bacterial STIs, Research and Development, Statens Serum Institut, Copenhagen, Denmark Received 4 February 2010/Returned for modification 31 March 2010/Accepted 12 August 2010
Mycoplasma genitalium is an established cause of sexually transmitted infections. Studies of disease associations are often performed on archived specimens, but little is known about the effect of storage of specimens on the detection of M. genitalium. Genital swab and first-void urine specimens submitted for detection of M. genitalium were tested on the day of receipt. Remnants of positive original specimens as well as DNA preparations were stored at ⴚ20°C for up to 18 months. A total of 361 M. genitalium-positive specimens were available. PCR after repeat DNA preparation was performed for 262 specimens. The sensitivity after repeat DNA preparation was 90%, and the median decrease in DNA load was 155 genome equivalents (geq) (P < 0.0001). For 327 specimens, PCR could be repeated on the primary DNA preparation. The sensitivity of PCR after storage was 95%, and the median decrease in DNA load was 13.5 geq (P < 0.0001). The specimens yielding negative results at repeat testing had a significantly lower median DNA load in the primary analysis than those with a repeat positive test (P < 0.0001). For 228 specimens, PCR could be performed both on the primary DNA preparation and after repeat DNA preparation. The median DNA load was lower after repeat DNA extraction than after repeat testing of the stored DNA extract (P < 0.0001). In conclusion, the M. genitalium DNA load as well as the detection rate decreased after storage. This was more pronounced in clinical specimens stored frozen than in stored DNA extracts, particularly in those with an initial low DNA load. Mycoplasma genitalium was first isolated in 1980 from 2 of 13 men with urethritis (17) and is now an established cause of nongonococcal urethritis (NGU) in both men and women (1, 4, 6, 11, 14). Urethritis is one of the most common conditions among men presenting at sexually transmitted disease (STD) clinics. In Scandinavia, the prevalence of gonococcal urethritis has drastically decreased during recent years, and consequently, most of the patients present with NGU. Chlamydia trachomatis is found in 20 to 35% of the NGU patients, and M. genitalium is the cause of disease in 20 to 35% of those with nonchlamydial NGU (NCNGU) (7). Furthermore, M. genitalium has been associated with cervicitis in most studies (1, 12), and there is increasing evidence that it may cause pelvic inflammatory disease (PID) (2, 3, 5, 16). Studies of disease associations are often performed on archived specimens, and the stability of the microbial nucleic acid may affect the detection rate. We aimed to determine the effect of freezing at ⫺20°C on the level of recovery of M. genitalium from stored clinical specimens compared to that from stored DNA extracts. (Parts of this study were presented at the 18th International Society for STD Research [ISSTDR] meeting in London, United Kingdom, June 2009.)
tubes; cervical, vaginal, and urethral swab specimens were collected and transported in a variety of transport media used for C. trachomatis testing, primarily ProbeTec medium (BD, Sparks, MD), Amplicor UTM (Roche Molecular Diagnostics, Pleasanton, CA), Copan Universal Transport Medium (UTM) (Copan, Brescia, Italy), and 2SP chlamydia transport medium (Statens Serum Institut [SSI], Hilleroed, Denmark). A few specimens were collected in Stuart’s transport medium (SSI). M. genitalium was detected by an inhibitor-controlled quantitative MgPa-gene TaqMan real-time PCR (9) on the day of receipt, and all positive results were subsequently confirmed using a conventional gel-based 16S rRNA gene PCR (10). The standard curve for the quantitative PCR was generated from 10-fold dilution series of M. genitalium genomic DNA ranging from 1 genome equivalent (geq) per l to 100,000 geq/l. All quantitative results were expressed as the number of geq/5 l template used in the PCR and were determined as the mean of two wells. The same stock of M. genitalium DNA was used to prepare the standards throughout the study. Sample preparation was performed essentially as described previously (8). In brief, 1.9 ml of the FVU specimens was centrifuged at 20,000 ⫻ g for 15 min, the pellet was resuspended in 300 l of a 20% Chelex 100 slurry (Bio-Rad, Hercules, CA) in TE (Tris-EDTA) buffer, and the suspension was incubated at 95°C for 10 min. From swab specimens collected in BD ProbeTec transport medium, 950 l was treated as described above for FVU specimens. For swab specimens collected in Roche STM, 2SP chlamydia transport, and Copan UTM medium, 100 l of the transport medium was aspirated and added directly to 300 l of Chelex slurry. For specimens collected in Stuart’s transport medium, the charcoal-impregnated cotton swab was placed in 1 ml of 2SP chlamydia transport medium and vortexed thoroughly, and 100 l of the medium was added directly to 300 l of Chelex slurry. Remnants of M. genitalium-positive original specimens as well as DNA preparations were stored at ⫺20°C for 1 to 18 months (median, 296 days). The freezers were standard commercial models without automatic defrosting, thus avoiding repeated, unintended freeze-thaw cycles. Furthermore, they were centrally temperature monitored in order to document uninterrupted function. Clinical specimens were subjected to repeat DNA preparation and quantitative PCR (repeat DNA), and the corresponding primary DNA preparations were thawed, vortexed, and briefly centrifuged before repeat quantitative PCR (repeat PCR). A total of 361 M. genitalium-positive specimens were received from July 2007 through January 2009; 166 were collected from 127 women and 195 were collected from 178 men. Figure 1 shows the number of specimens tested after storage under different conditions. From women, 25 FVU specimens, 33 urethral swab specimens, 92 cervical swab specimens, and 12 vaginal swab specimens were received. For four swab specimens, the anatomical site was not available. From men, 106 urethral swab specimens and 89 FVU specimens were examined.
MATERIALS AND METHODS Specimens with requests for diagnostic M. genitalium PCR testing were received from general practitioners, STD clinics, and private specialists. First-void urine (FVU) specimens were submitted in sterile polypropylene or polystyrene
* Corresponding author. Mailing address: Bacterial STIs, Research and Development, Statens Serum Institut, DK-2300 Copenhagen S, Denmark. Phone: 45 3268 3636. Fax: 45 3268 3152. E-mail:
[email protected]. 䌤 Published ahead of print on 18 August 2010. 3624
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M. GENITALIUM SPECIMEN STORAGE
FIG. 1. Flowchart showing numbers of specimens included and tested at different stages of storage independent of type of specimen.
Statistical analysis. Fisher’s exact test was used to test for differences in proportions; McNemar’s test was used to compare paired proportions, but the exact P value of McNemar’s test statistic, based on the binomial distribution, was used (Liddell’s test); the Mann-Whitney test was used to test for differences in continuous variables between groups; the Wilcoxon signed-ranks test was used to test for differences between matched pairs (i.e., the DNA loads before and after freezing). These tests were performed with the StatsDirect (version 2.6.6) program (StatsDirect Ltd., Cheshire, United Kingdom).
RESULTS Considering the DNA load on primary testing, the 166 specimens from women had a median DNA load of 25 geq. There was no difference in the median DNA loads in the 92 cervical swab specimens (median, 23 geq), the 23 urethral swab specimens (median, 25 geq), or the 25 FVU specimens (median, 38 geq). However, the 12 vaginal swab specimens contained a median of 107 geq, which was significantly higher than the DNA load in the cervical swab specimens (P ⫽ 0.046) but not significantly different from the loads in the other specimen types. Among the 195 specimens from men, the median DNA load for all specimens was 342 geq; FVU and urethral swab specimens had similar DNA loads (416 and 257 geq, respectively; P ⫽ 0.7). No differences in the DNA load could be found when the results for different transport media with similar specimen types were compared (data not shown). PCR could be performed again after repeat DNA preparation for 262 (73%) of the 361 specimens, comprising 166 specimens from 127 women (median age, 24 years; age range, 16 to 57 years) and 195 specimens from 178 men (median age, 27 years; age range, 15 to 63 years). After repeat DNA preparation and subsequent PCR, 25 were negative and the median decrease in the DNA load was 155 geq (P ⬍ 0.0001). Thus, the sensitivity of PCR after repeat DNA preparation was 90%, which was significantly lower than that of the primary analysis (P ⬍ 0.0001) (Table 1). The specimens yielding negative results in the primary analysis had a significantly lower median DNA load (4 geq) than specimens yielding a positive repeat test result (129 geq) (P ⬍ 0.0001). In order to estimate the dynamic change in sensitivity, the DNA load and sensitivity for specimens stored for the shortest and longest quartiles were compared. The 66 specimens stored for ⬍130 days (lowest quartile) had a median decrease in DNA load of 4.5 geq, and the sensitivity of repeat DNA extraction was 95% (63 of 66 specimens). In comparison, specimens stored for ⬎478 days (the highest quartile) had a median decrease of 39.5 geq (P ⫽ 0.013
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compared to the lowest quartile) and a sensitivity of 86% (57 of 66 specimens) (P ⫽ 0.079). Although the difference in sensitivity was not statistically significant, these findings suggest that the decrease in sensitivity caused by freezing may increase with the length of storage. For 327 (91%) of the 361 specimens, PCR could be repeated on the primary DNA preparation; of these, 15 were negative and those that were repeat negative had a lower median DNA load in the primary analysis (3.5 geq versus 107 geq for those that were repeat positive; P ⬍ 0.0001). The sensitivity of PCR performed after storage of the primary DNA preparation was 95%, which was significantly lower than that of the PCR performed after primary analysis (P ⬍ 0.0001). In 228 (63%) of the 361 specimens with an initial median DNA load of 78 geq, PCR could be repeated both on the primary DNA preparation and after repeat DNA preparation. For this group of specimens where test results were available for both storage conditions, repeat PCR on the primary DNA extract had a sensitivity of 94% and showed a median DNA load of 37 geq. PCR on the repeat DNA extract had a sensitivity of 89% and showed a median DNA load of 21 geq. The median DNA load was lower after repeat DNA extraction than after repeat testing of the stored DNA extract (median difference, 46 geq; P ⬍ 0.0001), and the sensitivity of testing after repeat DNA extraction was significantly lower than that after testing of the stored DNA preparation (P ⫽ 0.035). The results of primary PCR, repeat PCR, and repeat DNA, stratified by sex, specimen type, and the different transport media used, are presented in Table 1. DISCUSSION The present study aimed to quantify the effect of freezing at ⫺20°C on the detection of M. genitalium by PCR. We previously used a conventional gel-based PCR (10) to evaluate the effect of freezing on 102 M. genitalium-positive FVU specimens from males and 22 FVU specimens from females extracted by the Chelex method (repeat PCR) and found that 94% of the male FVU specimens and all of the female FVU specimens remained positive after storage (8). Similarly, repeat DNA preparation was performed for 68 of the corresponding original male FVU specimens and for 15 female FVU specimens which had been stored for 1 to 18 months at ⫺20°C. A sensitivity of 93% for the male FVU specimens was found after repeat DNA extraction, while only 73% of the female FVU specimens remained positive after the specimens were frozen. However, only FVU specimens were studied and the gel-based assay did not allow quantitation. In the present study, the sensitivities found for repeat PCR on male and female FVU specimens as well as the sensitivities for testing of repeat DNA were not statistically different from the results found previously, and since similar sample preparation methods were applied, pooling of the two data sets would provide a better estimate of the sensitivity. If this approach is followed, the sensitivity for repeat PCR on DNA extracted before freezing of male FVU specimens would be 96% (95% confidence interval [CI], 92 to 98%; 174/182 specimens), and for female FVU specimens the sensitivity would be 98% (95% CI, 88 to 99.9%; 42/43 specimens). For repeat DNA extraction, the sensitivity for male FVU specimens would be 92% (95% CI, 86 to 96%; 121/132 specimens) and that for female FVU specimens would be 83% (95% CI, 67 to
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TABLE 1. Results of primary PCR, repeat PCR, and repeat DNA analysisa
Stratification factor
Test 1
Test 2
No. of specimens
Median DNA load (geq) Test 1 Test 2
All specimens regardless of transport medium
Total, primary PCR Total, primary PCR Total, repeat DNA Female, primary PCR Female, primary PCR Female, repeat DNA Male, primary PCR Male, primary PCR Male, repeat DNA
Total, repeat DNA Total, repeat PCR Total, repeat PCR Female, repeat DNA Female, repeat PCR Female, repeat PCR Male, repeat DNA Male, repeat PCR Male, repeat PCR
262 327 228 124 149 107 138 178 121
78 86 21 24 25 7 417 353 100
21 50 37 7.5 13 10 102 279 327
Urine
Total urine, primary PCR Total urine, primary PCR Total urine, repeat DNA Female urine, primary PCR Female urine, primary PCR Female urine, repeat DNA Male urine, primary PCR Male urine, primary PCR Male urine, repeat DNA
Total urine, repeat DNA Total urine, repeat PCR Total urine, repeat PCR Female urine, repeat DNA Female urine, repeat PCR Female urine, repeat PCR Male urine, repeat DNA Male urine, repeat PCR Male urine, repeat PCR
85 101 73 21 21 17 64 80 56
99 163 21 31 38 7 276 386 179
21 116 46 12 19 14 62 421 60
BD-Probetec transport medium
Total, primary PCR Total, primary PCR Total, repeat DNA Female, primary PCR Female, primary PCR Female, repeat DNA Cervix, primary PCR Cervix, primary PCR Cervix, repeat DNA Female urethra, primary PCR Female urethra, primary PCR Female urethra, repeat DNA Male urethra primary PCR Male urethra, primary PCR Male urethra, repeat DNA
Total, repeat DNA Total, repeat PCR Total, repeat PCR Female, repeat DNA Female, repeat PCR Female, repeat PCR Cervix, repeat DNA Cervix, repeat PCR Cervix, repeat PCR Female urethra, repeat DNA Female urethra, repeat PCR Female urethra, repeat PCR Male urethra repeat DNA Male urethra, repeat PCR Male urethra, repeat PCR
103 98 94 46 42 40 28 26 25 13
243 246.5 90.5 33 39 16.5 30 33 16 142
84 143 160.5 16.5 15.5 15 16.5 15 14 18
2SP chlamydia transport medium
Female, primary PCR Female, primary PCR Female, repeat DNA
Female, repeat DNA Female, repeat PCR Female, repeat PCR
Copan UTM transport medium
Total, primary PCR Total, primary PCR Total, repeat DNA
Median difference in DNA load (geq 关95% CI兴))
P valueb
% sensitivity compared to primary PCR (P valuec)
154.5 (72 to 285) 13.5 (6 to 32) 46 (16 to 119) 13.5 (8 to 39) 10.5 (5 to 21) 3 (0 to 7) 804 (399 to 1,672) 23 (2 to 88) 504 (157 to 1,442)
⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 0.02 ⬍0.0001 0.02 ⬍0.0001
90 (⬍0.0001) 95 (⬍0.0001) NDd (0.035) 87 (⬍0.0001) 93 (0.01) ND (0.125) 93 (0.004) 98 (0.13) ND (0.3)
236.5 (57 to 898) 26 (2 to 100) 255.5 (17 to 964) 40 (4 to 138.5) 11 (⫺2 to ⫺98) 8 (⫺1 to ⫺49) 1,010 (154 to 2,974) 44 (0.5 to 483) 618 (126 to 2,584)
⬍0.0001 0.015 ⬍0.0001 0.008 0.08 0.10 ⬍0.0001 0.041 ⬍0.0001
91 (0.004) 97 (0.25) ND (0.13) 90 (0.5) 95 (⬎0.99) ND (⬎0.99) 91 (0.03) 98 (0.5) ND (0.38)
346 (141 to 771) ⬍0.0001 20 (3 to 101) 0.018 131 (37 to 507) ⬍0.0001 13 (2 to 44) 0.019 14.5 (6 to 34) 0.002 4.25 (⫺0.5 to 21) 0.092 12.5 (⫺0.5 to 32) 0.069 10 (⫺3 to 18) 0.094 2.5 (⫺4 to 16) 0.393 39.5 (⫺88 to 230) 0.168
11
243
10
20
55 54 53
1889 1823 469
469 677 736
19 17 17
10 7 7
8 10 10
44 (⫺4 to 388) 66 (0 to 462) 1 (⫺4 to 11)
0.196 0.035 0.64
Total, repeat DNA Total, repeat PCR Total, repeat PCR
8 9 8
204 248 86
86 66 137
138 (⫺13 to 413) 13 (⫺725 to 335) 71 (⫺26 to 1,064)
0.054 0.73 0.11
100 100 ND
Roche transport medium Total, primary PCR Total, primary PCR Total, repeat DNA
Total, repeat DNA Total, repeat PCR Total, repeat PCR
18 12 12
23.5 9.5 6
7.5 6 6
9 (⫺1 to 54) 7 (1 to 509) 4 (0 to 57)
0.12 0.021 0.078
100 92 (⬎0.99) ND (⬎0.99)
Stuart’s transport medium
Total, repeat DNA Total, repeat PCR Total, repeat PCR
21 21 19
9 9 4
4 5 5
11 (2 to 436) 6 (1 to 81) 1 (⫺2 to 3)
0.001 0.014 0.72
Total, primary PCR Total, primary PCR Total, repeat DNA
80
172 (21.5 to 342)
0.001
48.5
19 (⫺497 to 134)
0.193
93 (0.023) 96 (0.13) ND (0.25) 87 (0.03) 97 (⬎0.99) ND (0.25) 86 (0.13) 92 (0.5) ND (0.5) 96 (⬎0.99)
1,418 (694 to 5,761) ⬍0.0001 92 (⫺40 to 970) 0.212 868 (164 to 2,016) ⬍0.0001
100 ND (⬎0.99) 98 (⬎0.99) 98 (⬎0.99) ND (⬎0.99) 95 (⬎0.99) 88 (0.5) ND (⬎0.99)
76 (0.06) 86 (0.25) ND (0.5)
a Primary PCR consisted of sample preparation and PCR performed immediately after receipt of the specimens, repeat PCR consisted of sample preparation immediately after receipt of the specimen but PCR performed after storage at ⫺20°C, and repeat DNA consisted of initial storage of the clinical specimen at ⫺20°C, followed by subsequent DNA extraction and PCR. The results are stratified by sex, specimen type, and the different transport media used. Note that the number of specimens in each comparison may vary due to the lack of a complete data set for all specimens. b P value for difference in DNA load. c P value for difference in sensitivity. d ND, not determined.
94%; 30/36 specimens). The slightly lower sensitivity for repeat DNA extraction of female FVU specimens might be explained by the lower median number of genome copies in female specimens (31 geq for female FVU specimens compared to 276 geq for male FVU specimens). The present study was based on specimens submitted for detection of M. genitalium. Consequently, a wide range of different transport media were used; however, no significant
difference in the sensitivity after freezing and repeat DNA extraction could be found. This indicates that the decay of target DNA is largely independent of the transport medium and is an effect of the freezing alone. It is not surprising that M. genitalium cells may lyse after only one freeze-thaw cycle, and since clinical material may be rich in DNAses, the liberated DNA will be rapidly destroyed. Whether some of the newer transport media for FVU and swab specimens, such as Gene-
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Lock medium (Sierra Molecular Corporation) or Aptima transport medium (Gen-Probe Inc.), are more efficient in protecting the DNA remains to be determined. These transport media supposedly contain nucleic acid stabilizers and should provide less degradation. It is important, however, that the extraction procedure also extract liberated nucleic acid, and consequently, the centrifugation step included in the Chelex DNA extraction method may be less efficient than extraction procedures accommodating a large volume of urine or transport medium. Furthermore, since the length of storage without freeze-thaw cycles appears to have a significant effect on the decrease in DNA load, future studies should consider including storage at ⫺80°C for comparison. The findings of the present study are important for the interpretation of the findings of studies based on specimens that have been stored for a longer period of time at ⫺20°C. Manhart et al. recently estimated the prevalence of M. genitalium infection in a nationally representative sample of young adults in the United States using stored frozen urine specimens from 1,714 women and 1,218 men. The prevalence in women was found to be as low as 0.8% (13), but using the knowledge obtained from the present study, the true prevalence may actually have been closer to 1%. Similarly, the prevalence of M. genitalium in pregnant women was found to be 0.7% when it was determined with frozen stored urine specimens (15). If fresh material had been used, the prevalence would probably have been higher, in particular, when considering that the DNA load in asymptomatic subjects tends to be lower than that in symptomatic patients and that those specimens failing amplification after DNA extraction of frozen material were those with the lowest DNA loads. The decrease in sensitivity and DNA load should have implications for planning of future studies; DNA extraction should obviously be performed on fresh material, and if at all possible, testing should be performed before the DNA preparation is frozen. The present study has some limitations. No clinical information could be obtained, so a possible difference in sensitivity between symptomatic and asymptomatic patients could not be determined. Apparently, specimens form women contained a lower median number of genome copies than specimens from men, leading to a larger decrease in sensitivity for female specimens than for male specimens after storage. Whether this gender bias can be generalized or is an effect of a larger proportion of symptomatic men being tested would need further studies. Furthermore, the decrease in sensitivity may to some extent reflect a Poisson distribution leading to sampling error for specimens with a low concentration of template DNA. We did not perform repeat testing of frozen M. genitalium-negative specimens, but in a previous study (8), 2 out of 100 negative swab specimens were positive when they were tested with a concentration step consisting of centrifugation. When FVU specimens from these two patients were retested, they were actually M. genitalium positive, suggesting that some false-negative results should be expected with this rather crude DNA extraction procedure. In conclusion, freezing of clinical specimens as well as Chelex-extracted DNA leads to a significantly lower M. geni-
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talium DNA load and a decreased sensitivity compared to that obtained by testing of fresh specimens. The M. genitalium DNA load as well as the sensitivity decreased significantly more if the clinical specimen had been stored frozen at ⫺20°C than if the DNA had been extracted at the time of receipt of the specimen and the extract had been stored frozen. ACKNOWLEDGMENTS Birthe Dohn and Gitte Jensen are thanked for excellent technical assistance. The study was partially funded by Aage Bangs Fond and Civilingeniør Frode V. Nyegaard og Hustrus Fond. The corresponding author certifies, on behalf of both authors, that no manufacturer of a product discussed in the manuscript had a role, either directly or through a third party, in the gathering or preparation of data or in the writing of the manuscript. REFERENCES 1. Anagrius, C., B. Lore´, and J. S. Jensen. 2005. Mycoplasma genitalium: prevalence, clinical significance, and transmission. Sex. Transm. Infect. 81:458– 462. 2. Cohen, C. R., L. E. Manhart, E. A. Bukusi, S. Astete, R. C. Brunham, K. K. Holmes, S. K. Sinei, J. J. Bwayo, and P. A. Totten. 2002. Association between Mycoplasma genitalium and acute endometritis. Lancet 359:765–766. 3. Cohen, C. R., N. R. Mugo, S. G. Astete, R. Odondo, L. E. Manhart, J. A. Kiehlbauch, W. E. Stamm, P. G. Waiyaki, and P. A. Totten. 2005. Detection of Mycoplasma genitalium in women with laparoscopically diagnosed acute salpingitis. Sex. Transm. Infect. 81:463–466. 4. Falk, L., H. Fredlund, and J. S. Jensen. 2005. Signs and symptoms of urethritis and cervicitis among women with or without Mycoplasma genitalium or Chlamydia trachomatis infection. Sex. Transm. Infect. 81:73–78. 5. Haggerty, C. L., P. A. Totten, S. G. Astete, S. Lee, S. L. Hoferka, S. F. Kelsey, and R. B. Ness. 2008. Failure of cefoxitin and doxycycline to eradicate endometrial Mycoplasma genitalium and the consequence for clinical cure of pelvic inflammatory disease. Sex. Transm. Infect. 84:338–342. 6. Jensen, J. S. 2004. Mycoplasma genitalium: the aetiological agent of urethritis and other sexually transmitted diseases. J. Eur. Acad. Dermatol. Venereol. 18:1–11. 7. Jensen, J. S. 2006. Mycoplasma genitalium infections. Diagnosis, clinical aspects, and pathogenesis. Dan. Med. Bull. 53:1–27. 8. Jensen, J. S., E. Bjo ¨rnelius, B. Dohn, and P. Lidbrink. 2004. Comparison of first void urine and urogenital swab specimens for detection of Mycoplasma genitalium and Chlamydia trachomatis by polymerase chain reaction in patients attending a sexually transmitted disease clinic. Sex. Transm. Dis. 31: 499–507. 9. Jensen, J. S., E. Bjo ¨rnelius, B. Dohn, and P. Lidbrink. 2004. Use of TaqMan 5⬘ nuclease real-time PCR for quantitative detection of Mycoplasma genitalium DNA in males with and without urethritis who were attendees at a sexually transmitted disease clinic. J. Clin. Microbiol. 42:683–692. 10. Jensen, J. S., M. B. Borre, and B. Dohn. 2003. Detection of Mycoplasma genitalium by PCR amplification of the 16S rRNA gene. J. Clin. Microbiol. 41:261–266. 11. Jensen, J. S., R. Ørsum, B. Dohn, S. Uldum, A. M. Worm, and K. Lind. 1993. Mycoplasma genitalium: a cause of male urethritis? Genitourin. Med. 69: 265–269. 12. Manhart, L. E., C. W. Critchlow, K. K. Holmes, S. M. Dutro, D. A. Eschenbach, C. E. Stevens, and P. A. Totten. 2003. Mucopurulent cervicitis and Mycoplasma genitalium. J. Infect. Dis. 187:650–657. 13. Manhart, L. E., K. K. Holmes, J. P. Hughes, L. S. Houston, and P. A. Totten. 2007. Mycoplasma genitalium among young adults in the United States: an emerging sexually transmitted infection. Am. J. Public Health 97:1118–1125. 14. Moi, H., N. Reinton, and A. Moghaddam. 2009. Mycoplasma genitalium in women with lower genital tract inflammation. Sex. Transm. Infect. 85:10–14. 15. Oakeshott, P., P. Hay, D. Taylor-Robinson, S. Hay, B. Dohn, S. Kerry, and J. S. Jensen. 2004. Prevalence of Mycoplasma genitalium in early pregnancy and relationship between its presence and pregnancy outcome. BJOG 111: 1464–1467. 16. Simms, I., K. Eastick, H. Mallinson, K. Thomas, R. Gokhale, P. Hay, A. Herring, and P. A. Rogers. 2003. Associations between Mycoplasma genitalium, Chlamydia trachomatis, and pelvic inflammatory disease. Sex. Transm. Infect. 79:154–156. 17. Tully, J. G., D. Taylor-Robinson, R. M. Cole, and D. L. Rose. 1981. A newly discovered mycoplasma in the human urogenital tract. Lancet i:1288–1291.
