Comparison of Multiplex PCR Assay with Culture for Detection of Genital Mycoplasmas

Mycoplasma isolates and other bacterial strains.The following organisms were purchased from the American Type Culture Collection (ATCC): Ureaplasma urealyticum (ATCC 27618), M. genitalium (ATCC 33530), M. hominis (ATCC 23114), M. arthritidis (ATCC 14152), M. salivarium (ATCC 14277), M. fermentans (ATCC 15474), M. ovale (ATCC 23714D), M. penetrans (ATCC 55252), Acholeplasma oculi (ATCC 27350) Chlamydia trachomatis (ATCC VR-902B), Chlamydia pneumoniae (ATCC VR-1310), Candida albicans (ATCC 14000), Escherichia coli (ATCC 25922), Gardnerella vaginalis (ATCC A2508), Neisseria gonorrhoeae (ATCC 49981), Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 27336), Streptococcus pneumoniae (ATCC 27336), Streptococcus pyogenes (ATCC 19615), and Haemophilus influenzae (ATCC 9006). The following organisms were kindly provided by C. Chesa (New York State Department of Health, Albany, N.Y.): M. pneumoniae (ATCC 1428, ATCC 15531, ATCC 29342, and SP 300), M. hominis (ATCC 2331 and RRRB-NIH), and M. arthri (ATCC 14152). Isolates of Streptococcus agalactiae and the viridans streptococcus group were clinical isolates from Albany Medical Center.

Clinical specimens.All specimens received in the clinical laboratory for Ureaplasma culture from 85 patients seen at a fertility clinic between November 1998 and November 1999 were included in the present study. Specimens included 27 cervical swabs, 2 vaginal swabs, 4 female urine samples, 49 semen samples, 2 male urine samples and 1 nonspecified sample. Cervical and vaginal swabs were transported in 2 ml of 2SP medium (34). Urine samples were concentrated 10-fold by centrifugation for 30 min at 1,600 × g prior to testing. Specimens were cultured upon receipt, and the remaining material was frozen at −70°C for PCR testing. Analysis of these samples was done in compliance with federal and institutional review board policies.

Culture for genital mycoplasmas.Specimens were inoculated onto A7 agar (Becton Dickinson, Cockeysville, Md. 21030) and incubated at 37°C in 5% CO₂ for 5 days. Cultures were examined microscopically daily for 5 days for the appearance of typical mycoplasma colonies. A7 agar incorporates a direct test for urease that allows the differentiation of ureaplasma from the other Mycoplasmatales (34).

Multiplex PCR assay for genital mycoplasma infection.Bacterial DNA from 100 μl of specimen or transport media was isolated by lysis in 400 μl of NucliSens lysis buffer (Organon Teknika, Durham, N.C.), extracted with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), and extracted again with chloroform-isoamyl alcohol. DNA was then precipitated in 100% isopropanol, washed in 70% ethanol, and suspended in 15 μl of RNase-DNase free sterile deionized water (Sigma, St. Louis, Mo.). Multiplex PCR was performed with primers specific for highly conserved regions in the urease gene of Ureaplasma spp., the 140-kDa adhesion protein gene of M. genitalium, and the 16S rRNA gene of M. hominis (4, 5, 36). Hot-start PCR was performed on the equivalent of 25 μl of sample in 50-μl reactions containing a 0.2 mM concentration of deoxynucleoside triphosphate mixture, 10 mM Tris, 3 mM MgCl₂, 25 pmol of each unlabeled forward primers, and 25 pmol of biotin-labeled reverse primer (Table 1) (Synthetic Genetics, San Diego, Calif.) and 1.25 U of Gold Taq (Applied Biosystems, Foster City, Calif.). All reactions were performed in a GeneAmp PCR System 9600 Thermocycler (Perkin-Elmer, Norwalk, Conn.) under the following conditions: 1 cycle of 10 min at 95°C, followed by 35, two-step cycles of 95°C for 15 s and 60°C for 60 s, followed by 5 min at 72°C.

PCR product detection.Ureaplasma and M. genitalium PCR products were detected by enzyme-linked oligosorbent assay (ELOSA) as previously described (38). Briefly, 5 μl of heat-denatured biotinylated-PCR product was added to 100 μl of hybridization solution containing 1 pmol of horseradish peroxidase-labeled probe specific for the target region (Table 1) (Synthetic Genetics), 7.5× Denhardt solution, 3.5× SSPE, and 0.2 mg of herring sperm DNA (Invitrogen Corp., Carlsbad, Calif.)/ml in streptavidin-coated microtiter plates (Roche Molecular, Indianapolis, Ind.). Each probe was used in separate ELOSAs. Plates were incubated at 42°C for 60 min and washed six times with Coulter wash buffer (Coulter Corp., Miami, Fla.) in a Dynatech microtiter plate washer (Guernsey, United Kingdom). A color substrate, ortho-phenylenediamine (Abbott Diagnostics, Abbott Park, Ill.), was added to each well, followed by incubation at room temperature for 30 min before substrate development was stopped with 1 M H₂SO₄. The absorbance was measured at 490 nm, and data were analyzed by using a UVMax Microplate reader (Molecular Devices, Menlo Park, Calif.). Specimens were considered positive if the optical density at 490 (OD₄₉₀) was greater than the mean OD₄₉₀ of the negative amplification control plus a cutoff factor. The cutoff factor was derived from the mean OD₄₉₀ of 50 negative specimens plus two standard deviations. In addition, amplified products were visualized for specific fragment size bands (Fig. 1) under UV light after electrophoresis for 1 h at 100 V through a 7% acrylamide gel stained with ethidium bromide. PCR products with 334-bp bands, which are consistent with amplification of M. hominis, were further evaluated by digestion with NarI, which results in the digestion of M. hominis PCR product to fragments of 62 and 272 bp.

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FIG. 1.

Performance of multiplex PCR in mixed specimens. Amplification of DNA from 100 CFU of U. urealyticum, M. hominis, and M. genitalium, either individually or in combination, was carried out. The results are shown on a ethidium bromide-stained agarose gel, and the OD₄₉₀ was determined by ELOSA.

Analytical sensitivity.The analytical sensitivity was determined by amplification of twofold serial dilutions of bacterial DNA, either individually or as a mixture of all three organisms. Dilutions ranged from 3.13 to 100 CFU. The lower limit of detection (LOD) was the CFU equivalent of DNA in the last sample positive in the dilution series.

Expanded “gold standard” and resolution of discordant results.All samples were amplified in a second PCR targeting the multiple-banded antigen (MBA) gene of ureaplasma (41) under the conditions described above. Detection involved visualization on gel electrophoresis, which offered the additional benefit of speciation by amplicon size. U. parvum (formerly biovar 1 or parvo biovar) (31, 43) produces an amplicon of 403 bp, and U. urealyticum (biovar 2 or T960 biovar) produces an amplicon of 448 bp. Determination of a true-positive case was based on an expanded gold standard for infected patients, which was defined by a positive result by culture or by both amplification assays. Samples that were positive by culture and negative by PCR were investigated for the presence of inhibitors to amplification by PCR. These samples were spiked with ca. 50 CFU of U. urealyticum DNA and analyzed for DNA recovery by PCR.

Quality control.Published guidelines were followed to guard against nuclease and nucleic acid contamination (22, 26), including maintaining a strict physical separation between PCR setup and analysis, the use of sterile filter tips, and the regular decontamination of all surfaces with hypochlorite (27). Assay controls included a negative extraction control, derived from pooled negative specimens, and a negative amplification control, comprised of RNase-DNase-free sterile deionized water. Positive extraction and amplification controls were derived from the DNA equivalent of 125 CFU of ATCC strains of U. urealyticum, M. genitalium, and M. hominis.

Statistical analysis.Clinical data were evaluated to determine how many samples were positive by each test, as well as those positive by two or more tests and those positive by only one test. True positives for genital mycoplasma infection were patients with Mycoplasmatales detected by two or more tests. Sensitivity, specificity, positive and negative predictive values were calculated by Bayesian analyses. Confidence intervals were calculated by using the exact binomial method.

Analytical sensitivity and specificity of the multiplex PCR.This assay amplified and differentiated between Ureaplasma spp., M. hominis, and M. genitalium, whether present as individual organisms or as a mixture (Fig. 1). The analytical sensitivity was established by the amplification of serial dilutions of bacterial DNA in three separate experiments. The average LODs for each organism amplified individually was 10.8 CFU for U. urealyticum and M. genitalium and 8.8 CFU for M. hominis. When these organisms were mixed together, there was a minimal loss in sensitivity, with LODs of 12.5 CFU for U. urealyticum, 11.3 CFU for M. genitalium, and 10 CFU for M. hominis.

Multiplex amplifications were performed with ca. 500,000 CFU of M. arthritidis, M. salivarium, M. fermentans, M. ovale, M. penetrans, and Acholeplasma oculi, as well as 11 other urogenital and respiratory microorganisms. In all cases, no bands were visible on gel electrophoresis, and all yielded negative results by ELOSA, demonstrating that the multiplex PCR assay did not cross-react with other Mycoplasmatales or other common genitourinary and respiratory microorganisms tested.

Clinical specimens.Genital and urine specimens were obtained for culture of genital mycoplasma from 85 patients seen at a fertility clinic. The results of one cervical swab were excluded from analysis due to bacterial overgrowth in the mycoplasma culture. Of the 84 patient results analyzed, 21 were culture positive (25%). Seventeen culture-positive specimens were also multiplex PCR positive. An additional 11 specimens were PCR positive, but culture negative (PCR detection rate = 33%). Of the 21 culture-positive specimens, Ureaplasma spp. were isolated from 17 and Mycoplasma spp. were isolated from 4. Of the 28 PCR-positive specimens, 23 were positive for Ureaplasma spp., 3 were positive for M. hominis, and 2 were positive for both.

An analysis of test results according to specimen type demonstrated interesting differences in the performance of the two test systems (Table 2). In cervical or vaginal specimens, PCR detected mycoplasma in 15 of 28 specimens, whereas culture detected mycoplasma in only 8 specimens. In semen specimens, the culture was positive in 12 of 49 specimens, whereas PCR was positive in only 10 (only 8 of which were culture positive). In fact, the only specimens that were culture positive, PCR negative were these 4 semen specimens.

Resolution of discordant results.All discordant samples were at a minimum discrepant for ureaplasma. To determine true positive specimens, all samples were amplified with a set of primers targeting the MBA gene in the ureaplasma genome. Ureaplasma was detected in 23 specimens with the PCR for MBA. Ten appeared to be U. parvum, twelve were U. urealyticum, and one was mixed with both species. Patients were considered to have genital mycoplasmas if specimens were culture positive or positive for both amplification assays. Based on this definition, 30 patients were determined to be infected with genital mycoplasma, resulting in a prevalence rate of 36%. The multiplex PCR detected mycoplasmas in 26 true-positive specimens, as well as 2 specimens that were not confirmed positive (Table 3).

Thirteen semen specimens, including the four culture-positive, multiplex PCR-negative specimens, were analyzed for the presence of inhibitors of PCR. Samples were spiked with U. urealyticum DNA and amplified in the multiplex PCR. In all cases, the spiked DNA was recovered by amplification and detected by ELOSA. In fact, the OD₄₉₀ obtained from the PCR products of the spiked samples, compared to the values of the PCR product of the stock of DNA used for spiking, demonstrated complete recovery of DNA (data not shown). These results indicate that these semen specimens did not contain inhibitors of PCR.

Clinical sensitivity, specificity, and predictive values.After discrepant results were resolved, the sensitivity and specificity were determined to be 87 and 96%, respectively, for PCR. Hence, PCR improves test sensitivity by 24% compared to culture, which has a sensitivity of 70% (Table 4). Based on a prevalence of 36%, positive and negative predictive values of multiplex PCR were determined to be 94 and 93%, respectively, whereas the negative predictive value for culture was only 86%. These results demonstrated that the multiplex PCR enhances ureaplasma detection.