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Mycoplasma genitalium PCR Does Freezing of

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Mycoplasma genitalium PCR Does Freezing of

     JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2010, p. 36243627 Vol. 48, No. 10 0095-1137/10/$12.00 doi:10.1128/JCM.00232-10 Copyright ? 2010, American Society for Microbiology. All Rights Reserved.

    Mycoplasma genitalium PCR: Does Freezing of

    Specimens Affect Sensitivity?

    Katrina Hutton Carlsen and Jorgen Skov Jensen*

    Bacterial STIs, Research and Development, Statens Serum Institut, Copenhagen, Denmark

    Received 4 February 2010/Returned for modication 31 March 2010/Accepted 12 August 2010

Mycoplasma genitalium is an established cause of sexually transmitted infections. Studies of disease associ-

    ations 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 ;rst-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 prepa-

    rations were stored at 20?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 signi;cantly 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.

     tubes; cervical, vaginal, and urethral swab specimens were collected and trans- Mycoplasma genitalium was rst isolated in 1980 from 2 of 13 ported in a variety of transport media used for C. trachomatis testing, primarily men with urethritis (17) and is now an established cause of ProbeTec medium (BD, Sparks, MD), Amplicor UTM (Roche Molecular Diag- nongonococcal urethritis (NGU) in both men and women (1, 4, nostics, Pleasanton, CA), Copan Universal Transport Medium (UTM) (Copan, 6, 11, 14). Brescia, Italy), and 2SP chlamydia transport medium (Statens Serum Institut Urethritis is one of the most common conditions among men [SSI], Hilleroed, Denmark). A few specimens were collected in Stuarts transport medium (SSI). M. genitalium was detected by an inhibitor-controlled quantitative presenting at sexually transmitted disease (STD) clinics. In MgPa-gene TaqMan real-time PCR (9) on the day of receipt, and all positive Scandinavia, the prevalence of gonococcal urethritis has dras- results were subsequently conrmed using a conventional gel-based 16S rRNA tically decreased during recent years, and consequently, most gene PCR (10). The standard curve for the quantitative PCR was generated from of the patients present with NGU. Chlamydia trachomatis is 10-fold dilution series of M. genitalium genomic DNA ranging from 1 genome found in 20 to 35% of the NGU patients, and M. genitalium is 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 the cause of disease in 20 to 35% of those with nonchlamydial mean of two wells. The same stock of M. genitalium DNA was used to prepare the NGU (NCNGU) (7). Furthermore, M. genitalium has been standards throughout the study. Sample preparation was performed essentially associated with cervicitis in most studies (1, 12), and there is as described previously (8). In brief, 1.9 ml of the FVU specimens was centri- increasing evidence that it may cause pelvic inammatory dis- fuged 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 ease (PID) (2, 3, 5, 16). suspension was incubated at 95?C for 10 min. From swab specimens collected in Studies of disease associations are often performed on ar- BD ProbeTec transport medium, 950 l was treated as described above for FVU chived specimens, and the stability of the microbial nucleic specimens. For swab specimens collected in Roche STM, 2SP chlamydia trans- acid may affect the detection rate. We aimed to determine the port, and Copan UTM medium, 100 l of the transport medium was aspirated effect of freezing at 20?C on the level of recovery of M. and added directly to 300 l of Chelex slurry. For specimens collected in Stuarts transport medium, the charcoal-impregnated cotton swab was placed in 1 ml of genitalium from stored clinical specimens compared to that 2SP chlamydia transport medium and vortexed thoroughly, and 100 l of the from stored DNA extracts. medium was added directly to 300 l of Chelex slurry. Remnants of M. geni- (Parts of this study were presented at the 18th International talium-positive original specimens as well as DNA preparations were stored at Society for STD Research [ISSTDR] meeting in London, 20?C for 1 to 18 months (median, 296 days). The freezers were standard United Kingdom, June 2009.) commercial models without automatic defrosting, thus avoiding repeated, unin- tended freeze-thaw cycles. Furthermore, they were centrally temperature mon- itored in order to document uninterrupted function. Clinical specimens were MATERIALS AND METHODS subjected to repeat DNA preparation and quantitative PCR (repeat DNA), and Specimens with requests for diagnostic M. genitalium PCR testing were re- the corresponding primary DNA preparations were thawed, vortexed, and briey ceived from general practitioners, STD clinics, and private specialists. First-void centrifuged before repeat quantitative PCR (repeat PCR). urine (FVU) specimens were submitted in sterile polypropylene or polystyrene 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 col- lected from 178 men. Figure 1 shows the number of specimens tested after * Corresponding author. Mailing address: Bacterial STIs, Research storage under different conditions. From women, 25 FVU specimens, 33 urethral swab specimens, 92 cervical swab specimens, and 12 vaginal swab specimens were and Development, Statens Serum Institut, DK-2300 Copenhagen S, received. For four swab specimens, the anatomical site was not available. From Denmark. Phone: 45 3268 3636. Fax: 45 3268 3152. E-mail: jsj@ssi.dk. men, 106 urethral swab specimens and 89 FVU specimens were examined. Published ahead of print on 18 August 2010.

    3624

     VOL. 48, 2010 M. GENITALIUM SPECIMEN STORAGE 3625

     compared to the lowest quartile) and a sensitivity of 86% (57 of 66 specimens) (P 0.079). Although the difference in sen- sitivity was not statistically signicant, these ndings 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 signicantly lower than that of the PCR per- FIG. 1. Flowchart showing numbers of specimens included and formed after primary analysis (P 0.0001). tested at different stages of storage independent of type of specimen. 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. Statistical analysis. Fishers exact test was used to test for differences in For this group of specimens where test results were available proportions; McNemars test was used to compare paired proportions, but the exact P value of McNemars test statistic, based on the binomial distribution, was for both storage conditions, repeat PCR on the primary DNA used (Liddells test); the Mann-Whitney test was used to test for differences in extract had a sensitivity of 94% and showed a median DNA continuous variables between groups; the Wilcoxon signed-ranks test was used to load of 37 geq. PCR on the repeat DNA extract had a sensi- test for differences between matched pairs (i.e., the DNA loads before and after tivity of 89% and showed a median DNA load of 21 geq. The freezing). These tests were performed with the StatsDirect (version 2.6.6) pro- median DNA load was lower after repeat DNA extraction than gram (StatsDirect Ltd., Cheshire, United Kingdom).

    after repeat testing of the stored DNA extract (median differ-

    ence, 46 geq; P 0.0001), and the sensitivity of testing after RESULTS repeat DNA extraction was signicantly lower than that after

    Considering the DNA load on primary testing, the 166 spec- testing of the stored DNA preparation (P 0.035).

    The results of primary PCR, repeat PCR, and repeat DNA, imens from women had a median DNA load of 25 geq. There

    stratied by sex, specimen type, and the different transport was no difference in the median DNA loads in the 92 cervical

    media used, are presented in Table 1. swab specimens (median, 23 geq), the 23 urethral swab speci-

     mens (median, 25 geq), or the 25 FVU specimens (median, 38

    geq). However, the 12 vaginal swab specimens contained a DISCUSSION median of 107 geq, which was signicantly higher than the The present study aimed to quantify the effect of freezing at DNA load in the cervical swab specimens (P 0.046) but not

    20?C on the detection of M. genitalium by PCR. We previously signicantly different from the loads in the other specimen

    types. Among the 195 specimens from men, the median DNA used a conventional gel-based PCR (10) to evaluate the effect of load for all specimens was 342 geq; FVU and urethral swab freezing on 102 M. genitalium-positive FVU specimens from

    specimens had similar DNA loads (416 and 257 geq, respec- males and 22 FVU specimens from females extracted by the

    Chelex method (repeat PCR) and found that 94% of the male tively; P 0.7). No differences in the DNA load could be

    found when the results for different transport media with sim- FVU specimens and all of the female FVU specimens remained ilar specimen types were compared (data not shown). positive after storage (8). Similarly, repeat DNA preparation was PCR could be performed again after repeat DNA prepara- performed for 68 of the corresponding original male FVU spec- tion for 262 (73%) of the 361 specimens, comprising 166 spec- imens and for 15 female FVU specimens which had been stored imens from 127 women (median age, 24 years; age range, 16 to for 1 to 18 months at 20?C. A sensitivity of 93% for the male 57 years) and 195 specimens from 178 men (median age, 27 FVU specimens was found after repeat DNA extraction, while years; age range, 15 to 63 years). After repeat DNA prepara- only 73% of the female FVU specimens remained positive after tion and subsequent PCR, 25 were negative and the median the specimens were frozen. However, only FVU specimens were decrease in the DNA load was 155 geq (P 0.0001). Thus, the studied and the gel-based assay did not allow quantitation. In the sensitivity of PCR after repeat DNA preparation was 90%, present study, the sensitivities found for repeat PCR on male and which was signicantly lower than that of the primary analysis female FVU specimens as well as the sensitivities for testing of (P 0.0001) (Table 1). The specimens yielding negative results repeat DNA were not statistically different from the results found

    previously, and since similar sample preparation methods were in the primary analysis had a signicantly lower median DNA

    load (4 geq) than specimens yielding a positive repeat test applied, pooling of the two data sets would provide a better result (129 geq) (P 0.0001). In order to estimate the dynamic estimate of the sensitivity. If this approach is followed, the sensi- change in sensitivity, the DNA load and sensitivity for speci- tivity for repeat PCR on DNA extracted before freezing of male

    FVU specimens would be 96% (95% condence interval [CI], 92 mens stored for the shortest and longest quartiles were com-

    to 98%; 174/182 specimens), and for female FVU specimens the pared. The 66 specimens stored for 130 days (lowest quar-

    tile) had a median decrease in DNA load of 4.5 geq, and the sensitivity would be 98% (95% CI, 88 to 99.9%; 42/43 specimens). sensitivity of repeat DNA extraction was 95% (63 of 66 spec- For repeat DNA extraction, the sensitivity for male FVU speci- imens). In comparison, specimens stored for 478 days (the mens would be 92% (95% CI, 86 to 96%; 121/132 specimens) and

    that for female FVU specimens would be 83% (95% CI, 67 to highest quartile) had a median decrease of 39.5 geq (P 0.013

     3626 CARLSEN AND JENSEN J. CLIN. MICROBIOL.

     aTABLE 1. Results of primary PCR, repeat PCR, and repeat DNA analysis

     % sensitivity Median DNA Median difference No. of bcompared to load (geq) Stratication factor Test 1 Test 2 P value in DNA load specimens primary (geq 95% CI )) cTest 1 Test 2 PCR (P value) All specimens regardless Total, primary PCR 154.5 (72 to 285) 0.0001 90 ( 0.0001) Total, repeat DNA 262 78 21 of transport medium Total, primary PCR 13.5 (6 to 32) 0.0001 95 ( 0.0001) Total, repeat PCR 327 86 50 dTotal, repeat DNA 46 (16 to 119) 0.0001 ND(0.035) Total, repeat PCR 228 21 37 13.5 (8 to 39) 0.0001 87 ( 0.0001) Female, primary PCR Female, repeat DNA 124 24 7.5 Female, primary PCR 10.5 (5 to 21) 0.0001 93 (0.01) Female, repeat PCR 149 25 13 Female, repeat DNA Female, repeat PCR 107 7 10 3 (0 to 7) 0.02 ND (0.125) Male, primary PCR Male, repeat DNA 138 417 102 804 (399 to 1,672) 0.0001 93 (0.004) Male, primary PCR Male, repeat PCR 178 353 279 23 (2 to 88) 0.02 98 (0.13) Male, repeat DNA Male, repeat PCR 121 100 327 504 (157 to 1,442) 0.0001 ND (0.3) Urine Total urine, primary PCR Total urine, repeat DNA 85 99 21 236.5 (57 to 898) 0.0001 91 (0.004) Total urine, primary PCR Total urine, repeat PCR 101 163 116 26 (2 to 100) 0.015 97 (0.25) Total urine, repeat DNA Total urine, repeat PCR 73 21 46 0.0001 ND (0.13) 255.5 (17 to 964) Female urine, primary PCR Female urine, repeat DNA 21 31 12 40 (4 to 138.5) 0.008 90 (0.5) Female urine, primary PCR Female urine, repeat PCR 21 38 19 11 ( 2 to 98) 0.08 95 ( 0.99) Female urine, repeat DNA Female urine, repeat PCR 17 7 14 8 ( 1 to 49) 0.10 ND ( 0.99) Male urine, primary PCR Male urine, repeat DNA 64 276 62 1,010 (154 to 2,974) 0.0001 91 (0.03) Male urine, primary PCR Male urine, repeat PCR 80 386 421 44 (0.5 to 483) 0.041 98 (0.5) Male urine, repeat DNA Male urine, repeat PCR 56 179 60 618 (126 to 2,584) 0.0001 ND (0.38) BD-Probetec transport Total, primary PCR Total, repeat DNA 103 243 84 346 (141 to 771) 0.0001 93 (0.023) medium Total, primary PCR Total, repeat PCR 98 246.5 143 20 (3 to 101) 0.018 96 (0.13) Total, repeat DNA Total, repeat PCR 94 90.5 160.5 131 (37 to 507) 0.0001 ND (0.25) Female, primary PCR Female, repeat DNA 46 33 16.5 13 (2 to 44) 0.019 87 (0.03) Female, primary PCR Female, repeat PCR 42 39 15.5 14.5 (6 to 34) 0.002 97 ( 0.99) Female, repeat DNA Female, repeat PCR 40 16.5 15 4.25 ( 0.5 to 21) 0.092 ND (0.25) Cervix, primary PCR Cervix, repeat DNA 28 30 16.5 12.5 ( 0.5 to 32) 0.069 86 (0.13) Cervix, primary PCR Cervix, repeat PCR 26 33 15 10 ( 3 to 18) 0.094 92 (0.5) Cervix, repeat DNA Cervix, repeat PCR 25 16 14 2.5 ( 4 to 16) 0.393 ND (0.5) Female urethra, primary Female urethra, repeat 13 142 18 39.5 ( 88 to 230) 0.168 96 ( 0.99) PCR DNA Female urethra, primary Female urethra, repeat 11 243 80 172 (21.5 to 342) 0.001 100 PCR PCR Female urethra, repeat Female urethra, repeat 10 20 48.5 19 ( 497 to 134) 0.193 ND ( 0.99) DNA PCR Male urethra primary PCR Male urethra repeat DNA 55 1889 469 1,418 (694 to 5,761) 0.0001 98 ( 0.99) Male urethra, primary PCR Male urethra, repeat PCR 54 1823 677 92 ( 40 to 970) 0.212 98 ( 0.99) Male urethra, repeat DNA Male urethra, repeat PCR 53 469 736 868 (164 to 2,016) 0.0001 ND ( 0.99) 2SP chlamydia transport Female, primary PCR Female, repeat DNA 19 10 8 44 ( 4 to 388) 0.196 95 ( 0.99) medium Female, primary PCR Female, repeat PCR 17 7 10 66 (0 to 462) 0.035 88 (0.5) Female, repeat DNA Female, repeat PCR 17 7 10 1 ( 4 to 11) 0.64 ND ( 0.99) Copan UTM transport Total, primary PCR Total, repeat DNA 8 204 86 138 ( 13 to 413) 0.054 100 medium Total, primary PCR Total, repeat PCR 9 248 66 13 ( 725 to 335) 0.73 100 Total, repeat DNA Total, repeat PCR 8 86 137 71 ( 26 to 1,064) 0.11 ND Roche transport medium Total, primary PCR Total, repeat DNA 18 23.5 7.5 9 ( 1 to 54) 0.12 100 Total, primary PCR 7 (1 to 509) 0.021 92 ( 0.99) Total, repeat PCR 12 9.5 6 Total, repeat DNA Total, repeat PCR 12 6 6 4 (0 to 57) 0.078 ND ( 0.99) Stuarts transport Total, primary PCR Total, repeat DNA 21 9 4 11 (2 to 436) 0.001 76 (0.06) medium Total, primary PCR Total, repeat PCR 21 9 5 6 (1 to 81) 0.014 86 (0.25) Total, repeat DNA Total, repeat PCR 19 4 5 1 ( 2 to 3) 0.72 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 stratied 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. bvalue for difference in DNA load. Pcvalue for difference in sensitivity. Pd ND, not determined.

94%; 30/36 specimens). The slightly lower sensitivity for repeat difference in the sensitivity after freezing and repeat DNA

    extraction could be found. This indicates that the decay of DNA extraction of female FVU specimens might be explained by

    target DNA is largely independent of the transport medium the lower median number of genome copies in female specimens

    and is an effect of the freezing alone. It is not surprising that M. (31 geq for female FVU specimens compared to 276 geq for male

    genitalium cells may lyse after only one freeze-thaw cycle, and FVU specimens).

    since clinical material may be rich in DNAses, the liberated The present study was based on specimens submitted for

    DNA will be rapidly destroyed. Whether some of the newer detection of M. genitalium. Consequently, a wide range of

    different transport media were used; however, no signicant transport media for FVU and swab specimens, such as Gene-

     VOL. 48, 2010 M. GENITALIUM SPECIMEN STORAGE 3627

    Lock medium (Sierra Molecular Corporation) or Aptima talium DNA load and a decreased sensitivity compared to that

    obtained by testing of fresh specimens. The M. genitalium transport medium (Gen-Probe Inc.), are more efcient in pro-

    DNA load as well as the sensitivity decreased signicantly tecting the DNA remains to be determined. These transport

    more if the clinical specimen had been stored frozen at 20?C media supposedly contain nucleic acid stabilizers and should

    provide less degradation. It is important, however, that the than if the DNA had been extracted at the time of receipt of

    the specimen and the extract had been stored frozen. extraction procedure also extract liberated nucleic acid, and

     consequently, the centrifugation step included in the Chelex

    DNA extraction method may be less efcient than extraction ACKNOWLEDGMENTS procedures accommodating a large volume of urine or trans- Birthe Dohn and Gitte Jensen are thanked for excellent technical port medium. Furthermore, since the length of storage without assistance. freeze-thaw cycles appears to have a signicant effect on the The study was partially funded by Aage Bangs Fond and Civilin- decrease in DNA load, future studies should consider includ- geniør Frode V. Nyegaard og Hustrus Fond. ing storage at 80?C for comparison. The corresponding author certies, on behalf of both authors, that no manufacturer of a product discussed in the manuscript had a role, The ndings of the present study are important for the either directly or through a third party, in the gathering or preparation interpretation of the ndings of studies based on specimens of data or in the writing of the manuscript. that have been stored for a longer period of time at 20?C. Manhart et al. recently estimated the prevalence of M. geni- REFERENCES talium infection in a nationally representative sample of young 1. Anagrius, C., B. Lore?, and J. S. Jensen. 2005. Mycoplasma genitalium: prev- adults in the United States using stored frozen urine specimens alence, clinical signicance, and transmission. Sex. Transm. Infect. 81:458 from 1,714 women and 1,218 men. The prevalence in women 462. 2. Cohen, C. R., L. E. Manhart, E. A. Bukusi, S. Astete, R. C. Brunham, K. K. was found to be as low as 0.8% (13), but using the knowledge Holmes, S. K. Sinei, J. J. Bwayo, and P. A. Totten. 2002. Association between obtained from the present study, the true prevalence may ac- Mycoplasma genitalium and acute endometritis. Lancet 359:765766. 3. Cohen, C. R., N. R. Mugo, S. G. Astete, R. Odondo, L. E. Manhart, J. A. tually have been closer to 1%. Similarly, the prevalence of M. Kiehlbauch, W. E. Stamm, P. G. Waiyaki, and P. A. Totten. 2005. 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