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1,3,4Department of Clinical Microbiology1, Outpatient Sexually Transmitted Disease Clinic, Department of Dermatovenereology3, and Clinical Research Centre4, Örebro University Hospital, SE-70185 Örebro, Sweden 2Mycoplasma Laboratory, Department of Respiratory Infections, Meningitis and Sexually Transmitted Infections, Statens Serum Institut, DK-2300 Copenhagen, Denmark
Correspondence Margaretha Jurstrand margaretha.jurstrand{at}orebroll.se
Received May 5, 2004
Accepted August 30, 2004
A real-time LightCycler PCR (LC-PCR) with hybridization probes for detection of Mycoplasma genitalium in endocervical and first void urine specimens was developed and compared to a conventional PCR. The primers for both assays were identical and designed to amplify a 427 bp fragment of the 16S rRNA gene of M. genitalium. The LC-PCR assay had a detection limit of < 5 bacterial genomes per reaction when dilutions of genomic DNA from a type strain of M. genitalium were tested. First void urine from 398 men and first void urine and endocervical specimens from 301 women attending an STD clinic were analysed by LC-PCR and by the conventional PCR. Using the conventional PCR as reference, the LC-PCR had a specificity of 99.7 % and a sensitivity of 72.2 % for the detection of M. genitalium in first void urine samples from men. There was no significant difference in the performance of the LC-PCR assay compared to the conventional PCR when endocervical swabs were considered (58 and 65 %, respectively) or with a set of endocervical swab/urine specimens for which the LC-PCR assay detected 73 % of the infections (specificity = 98.6 % and sensitivity = 68.2 %) while the conventional PCR detected 85 % of the infections. With female urine specimens there was a significant difference between the two assays (38 and 73 %, respectively; P = 0.01 McNemar's test). This illustrates the need to analyse both endocervical and urine specimens, because M. genitalium DNA was detected in only one of the two specimens in a great number of the M. genitalium-infected women. The lower sensitivity of the LC-PCR assay was probably caused by a combination of inhibition and limitations regarding the amount of template DNA. The LC-PCR assay was easy to perform and the simultaneous amplification and detection eliminated the need for further handling of PCR products. With improvement in sample preparation methods and increased volumes of the template DNA, the LC-PCR assay could be a useful routine diagnostic method.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Due to a lack of reliable culture and serological methods, the role of M. genitalium in NCNGU has been difficult to establish (Jensen et al., 1993; Taylor-Robinson, 1983), but the progress of molecular techniques like PCR has made it possible to detect the bacterium in urogenital specimens. PCR-based assays have been developed by several research groups but most of them are labour intensive and none is commercially available (Deguchi et al., 2002; Jensen et al., 1991; Taylor-Robinson, 1995).
The purpose of the present study was to further develop an existing conventional PCR protocol into a real-time PCR with hybridization probes for detection of M. genitalium DNA, and to evaluate it as a method of detecting M. genitalium in first void urine and endocervical specimens.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Endocervical specimens were obtained from females (n = 321), using four sterile Dacron swabs. The first and second swabs were placed into one polypropylene tube (Sarstedt) and the third and fourth swabs into another tube, both containing 2 ml 2SP medium (sucrose-phosphate buffer; 5 %, v/v, fetal bovine serum; and antibiotics). The specimens were randomly (by a dice) assigned to be sent to SSI or directly transported to UHÖ and stored at –70 °C until used for isolation of DNA to detect M. genitalium. The first void urine samples (10–20 ml) were collected at the clinical examination and divided into two sterile screw-cap polypropylene tubes (Sarstedt): one was sent to SSI and the other was sent to UHÖ. Likewise, first void urine samples were obtained from men attending the STD clinic.
If the patients were found to be positive for M. genitalium they were treated with antibiotics (azithromycin or tetracycline) and recalled for a check-up visit 4–5 weeks after the initial sample was obtained. These specimens were not included in the evaluation.
The first 314 urine samples were stored for 1–3 days at 2–8 °C at UHÖ until extraction of DNA to detect M. genitalium was performed, while the remaining specimens were stored at –20 °C before DNA was extracted. At SSI sample preparation was performed without previous freezing. Most often, the specimens were tested on the day of receipt, otherwise pre-treated specimens were stored at –20 °C until analysed in the PCR assay.
Sample preparation.
At UHÖ, DNA was released from the clinical specimens using Chelex 100 resin (Bio-Rad) (Walsh et al., 1991). A volume of 100 µl from urogenital specimens (in 2SP medium) was added to 1000 µl saline, or 1800 µl urine was directly transferred to Eppendorf tubes and microcentrifuged for 15 min at 20 000 g. The pellet was resuspended in 300 µl 5 % (w/v) Chelex 100 resin in distilled water, thoroughly mixed and incubated at 99 °C for 10 min, mixed again and the cell debris was pelleted by centrifugation at 12 000 g for 5 min. The supernatant containing the DNA was used in the real-time PCR. At SSI, 100 µl of the swab specimen was added directly to 300 µl of a 20 % (w/v) Chelex 100 slurry in TE buffer (pH 8.0) or the pellet from the first void urine specimens was resuspended in the same volume as previously described (Jensen et al., 2003).
LC-PCR.
The real-time PCR, with hybridization probes, was performed in a LC-PCR system (Roche Molecular Biochemicals).
The MG16-45F/MG16-447R primer set used for conventional PCR (Jensen et al., 2003) was used together with sequence-specific oligonucleotide probes labelled with fluorescent dyes. The sequences of the two probes were designed such that they hybridized to the amplified DNA fragment in a head to tail arrangement, separated by one nucleotide.
The primers and probes in this LC-PCR assay were designed by J. S. Jensen as a further development of the conventional PCR (Jensen et al., 2003) to detect a 427 bp fragment of the 16S rRNA gene sequence of the M. genitalium G-37 type strain (accession no. X77334). The reaction mix contained 2 µl FastStart DNA Master Hybridization Probes, containing FastStart Taq DNA polymerase, reaction buffer, dNTP mix (with dUTP instead of dTTP) and 10 mM MgCl2 (Roche Diagnostics). In the optimized PCR, MgCl2 was added to a final concentration of 5 mM and 0.6 µM of the forward primer MG16-45F (5'-TAC ATG CAA GTC GAT CGG AAG TAG C-3') and 0.4 µM of the reverse primer MG16-447R (5'-AAA CTC CAG CCA TTG CCT GCT AG-3') was used. The primers were purchased from Scandinavian Gene Synthesis (SGS). The two hybridization probes Mg16S-137-probe (LC-red 640 AAT TCA TGC GAA CTA AAG TTC TTA TGC GGT ATT AGC T – phosphate) and Mg16S-169-probe (AAT AAC GAA CCC TTG CAG GTC CTT TCA ACT T – fluorescein) (TIB MOLBIOL; Syntheselabor) were used at a final concentration of 0.2 µM each. The reaction mix (18 µl) was added to the glass capillary together with 2 µl template DNA from the clinical specimen. Included in each run was a positive M. genitalium control and water as a negative control.
The PCR program started with a pre-incubation for activation of the FastStart enzyme at 95 °C for 10 min, followed by amplification for 45 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 10 s, and extension at 72 °C for 16 s, and at the end of the protocol a cooling program (40 °C for 30 s) was set. Fluorescence was measured by the LightCycler instrument's fluorimeter at the end of each annealing step for the real-time detection.
The detection limit of the assay was determined by subjecting a 10-fold dilution series of genomic DNA from M. genitalium G-37T to the LC-PCR assay. The most concentrated DNA dilution contained 105 genomes ml–1. Two microlitres of each dilution (containing 105–100 genomes ml–1) was analysed in the LC-PCR reaction.
Conventional PCR for verification.
At UHÖ a positive result from the LC-PCR was verified by a conventional PCR with the same primers as the method used at SSI (Jensen et al., 2003) but with minor modifications: 1.5 U AmpliTaqGold DNA Polymerase (PE Biosystems), 1x PCR buffer (PE), 2.5 mM MgCl2, 200 µM of each dNTP (PE) and 0.4 µM of each primer (SGS). Also included in the reaction mix was an internal process control (Jensen et al., 2003). The amplification product was visualized after electrophoresis through a 2 % agarose gel containing ethidium bromide. At SSI, all positive results were confirmed by an independent PCR amplifying a fragment of the MgPa gene as previously described (Jensen et al., 2003). After comparing the results between UHÖ and SSI all discrepant specimens were re-analysed, and if inhibition was found (using the internal process control), they were re-tested with different amounts of template DNA (10, 5 and 2 µl) in the conventional PCR, and diluted 1/5 and 1/10 when re-tested in the LC-PCR assay.
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RESULTS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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A total of 19 (4.8 %) of 398 men were found to be positive for M. genitalium in one or both PCR methods when the first pass urine specimens were analysed (Table 1). Six men were still M. genitalium-positive at follow-up visit after initial antibiotic (tetracycline) treatment but are not included in the evaluation. As shown in Table 2, the LC-PCR assay detected 14 specimens M. genitalium DNA positive while 18 specimens were found to be M. genitalium DNA positive by the conventional PCR assay. Using the conventional PCR method as the ‘gold standard', the sensitivity of the LC-PCR assay was 72.2 % with a 95 % confidence interval (CI) of 46.5–90.3 %, and the specificity was 99.7 % (95 % CI = 98.5–99.9 %). There were five specimens tested positive at SSI that were missed by the LC-PCR, but when re-tested, two were positive (see Tables 1 and 2). In the evaluation of sensitivity these two specimens were considered LC-PCR negative. One sample was negative at SSI but positive in the LC-PCR assay.
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Considering the 19 men true positives when one or both PCR assays were M. genitalium DNA positive, the LC-PCR assay detected 14 (74 %) of the infections whereas the conventional PCR at SSI detected 18 (95 %), but the difference was not significant (P = 0.2 McNemar's test).
First void urine and endocervical specimens from 301 women were analysed, and 26 women were found to be M. genitalium DNA positive in either the swab or the urine specimen by the LC-PCR assay and/or the conventional PCR assay at SSI (Table 3). As shown in Table 4, the LC-PCR assay detected 19 of the 26 (73 %) women deemed M. genitalium positive and the conventional PCR assay detected 22 (85 %), not counting the follow-up visit specimens. Consequently, the sensitivity of the LC-PCR assay was 68.2 % (95 % CI = 45.1–86.1 %) and the specificity was 98.6 % (95 % CI = 96.4–99.6 %), when using the conventional PCR method as the ‘gold standard'. When the specimens were re-tested one more specimen was found to be positive in each laboratory.
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As shown in Table 4, the LC-PCR assay performed better with the endocervical swab specimens than with the female first void urine specimens. Considering the 26 women with at least one positive M. genitalium PCR result as true positives, the LC-PCR performed on urine specimens detected only 10 (38 %) of the infections whereas the conventional PCR detected 19 (73 %) of the infections (P = 0.01 McNemar's test). No significant difference was found between the performances of the two assays when endocervical swab specimens were considered: the LC-PCR detected 15 (58 %) of the infected women as compared to 17 (65 %) detected with the conventional PCR.
As described above, the order of sampling of the endocervical specimens was random. Three women had negative M. genitalium specimens in the LC-PCR assay but positive in the conventional PCR when the first swab specimens were sent to SSI, and two of the three women were M. genitalium-positive in the urine specimen, and the patients were hence assessed to be positive at UHÖ. In contrast, two specimens were positive by LC-PCR but negative in the conventional PCR although the first swab specimen was sent to SSI. Five women were found to be M. genitalium-positive at follow-up visit after antibiotic treatment of their initial infection, but are not included in the evaluation. The prevalence of M. genitalium-infected women in this population was found to be 8.6 % (26/301).
The LC-PCR positive specimens were re-tested once and also verified at UHÖ by using the conventional PCR assay used at SSI with minor modifications. During the LC-PCR analysis of the discrepant specimens, 10 urine specimens were inhibited (from eight women and two men), using the internal process control in the conventional PCR for verification (Tables 1 and 3). When the specimens were diluted and re-tested, M. genitalium DNA could not be detected.
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DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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In order to evaluate the LC-PCR assay with clinical material, specimens from men and women attending the STD clinic were analysed at the local laboratory by the LC-PCR assay and also sent to the Mycoplasma Laboratory in Copenhagen, where the conventional PCR was well established. Both laboratories applied confirmatory testing of positive results and, although different approaches were chosen, we decided to consider a patient M. genitalium-infected if only one specimen was confirmed positive in one of the laboratories. Using this criterion, 19 men and 26 women were considered M. genitalium-infected (Tables 1 and 3). When looking at the performance of the LC-PCR assay compared to the conventional PCR, there were no significant differences when endocervical swabs were considered (58 and 65 %, respectively) or female sets of endocervical swab/urine specimens, with which the LC-PCR assay detected 73 % (19/26) while the conventional PCR detected 85 % (22/26). This illustrates the need to analyse both specimens, because in a great number of M. genitalium-infected women M. genitalium DNA was detected only in one of the two specimens. The LC-PCR assay had a specificity of 99.7 % for men and 98.6 % for women, when compared with the conventional PCR.
Although the urine specimens were divided into equal parts after collection and thus were expected to yield the most comparable results, the sensitivity was significantly different between the two assays when female urine specimens were tested. For both men and women, the LC-PCR assay detected fewer infections than did the conventional PCR. The most likely explanation for this finding is the presence of inhibitors often found in urine as well as the lower input of template DNA in the LC-PCR assay. When analysing the LC-PCR-negative discrepant urine specimens, an inhibition of the internal process control was noticed in a substantial proportion of specimens in the gel-based PCR. The most frequent inhibitions were found in female urine specimens as previously described (Chernesky et al., 1997). Inhibitors in the urine specimens together with a small volume of template DNA in the LC-PCR assay led to a sensitivity of 42 % while the sensitivity when analysing endocervical swabs was found to be 65 % (Table 4). Other studies have found inhibitory activities when assaying M. genitalium by PCR (Casin et al., 2002; Chernesky et al., 1997; Jensen et al., 2003) and, therefore, we used sample dilution (1/5 and 1/10) in the LC-PCR assay, but M. genitalium DNA could not be detected in the diluted samples. The LC-PCR assay only uses 2 µl DNA in each capillary, thus it is likely that the DNA load was too low in these samples. Large differences in the bacterial load were described by Yoshida et al. (2002), who developed a real-time PCR assay for quantification of M. genitalium, and recently, using a quantitative TaqMan assay, it was demonstrated that 14 % of male first void urine specimens contained less than 2 genome copies (µl pre-treated specimen)–1 using a similar sample preparation method to that of the current study (Jensen et al., 2004). Together with the inhibition problem, this led to a lower sensitivity in the LC-PCR compared to the conventional PCR, which uses an input of 10 µl template DNA and an internal control for inhibition.
The endocervical specimens were randomized to control for a possible influence on the performance relating to the sequence of obtaining the specimen. However, no trend was observed towards demonstrating a difference depending on sampling sequence. It is possible that some of the differences in results could be due to the time of transportation. However, since the overall performance of the conventional PCR was superior to that of the LC-PCR assay, which had the shortest transport time, this explanation seems less likely. It should be noted, however, that a large proportion of the urine specimens received at UHÖ had been examined after storage at –20 °C. It could be suspected that freezing may lyse the fragile M. genitalium cells and expose their DNA to Dnases, which are present in high concentrations in urine, and thus lead to false negative results in the LC-PCR. On the contrary, there is some evidence (Berg et al., 1997) that freezing and thawing of urine specimens is one way of removing inhibitors, which should theoretically enhance the sensitivity.
Although patients found to be M. genitalium DNA positive were treated with standard antibiotic treatment (tetracyclines) used for urethritis and cervicitis, four women and six men were still M. genitalium-positive at the follow-up visit. This demonstrates that tetracyclines are inappropriate for treatment of M. genitalium infections (Falk et al., 2003), and strengthens the evidence that M. genitalium is associated with persistent or recurrent infection among both women and men (Björnelius et al., 2000; Falk et al., 2003; Maeda et al., 2001; Uuskula & Kohl, 2002).
It is increasingly evident that M. genitalium may cause NGU in men (Jensen, 2004), and several studies suggest that it may cause urethritis and cervicitis in women (Anagrius & Lore, 2002; Casin et al., 2002; Cohen et al., 2002; Uno et al., 1997). The aim of the present study was not to evaluate signs and symptoms among the M. genitalium-infected, but the findings in this study show that M. genitalium is frequently encountered in the genital tract of female patients attending an STD clinic.
In conclusion, this real-time LC-PCR method using hybridization probes provided a specific method for detection of M. genitalium. It has some advantages over conventional PCR assays, which are labour-intensive and generally more prone to contamination. The LC-PCR assay is easy to perform and the simultaneous amplification and detection eliminates the need for further handling of PCR products and decreases the risk of contamination. However, in order to implement this method for routine diagnostics an internal amplification control should be incorporated and the sample preparation method should be improved to minimize the inhibition problems. The low load of M. genitalium DNA in many specimens necessitates a concentration step or larger template DNA volumes to be subjected to the PCR to increase the sensitivity. To our knowledge, this is the first interlaboratory evaluation of M. genitalium PCR assays, incorporating a large number of prospectively collected urogenital specimens, that demonstrates that despite an apparent excellent sensitivity with purified DNA the clinical performance may vary considerably.
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ACKNOWLEDGEMENTS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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