Design
This study describes the results of immunoblot and ELISA assays for the detection of M. genitalium antibodies in serum and of PCR for the detection of M. genitalium DNA in cervical and vaginal swabs to determine their usefulness over time as diagnostic tools to identify M. genitalium culture-positive women.

Participants
A subpopulation of 31 low-income minority women from whom positive M. genitalium cultures were established, who were enrolled in a randomized controlled trial of behavioral-cognitive interventions to reduce the recurrence of STDs in San Antonio, TX, were tested. Stored serum samples from 54 pregnant women with no known history of STDs were used as negative controls for the antibody detection assays. Samples were collected from each woman at each clinic visit over a period of 17 to 39 months. The number of visits with PCR data ranged from 3 to 12, with an average of 6.9. Two hundred serum samples were tested.

Description of Tests and Diagnostic Standard
Cervical and vaginal swab specimens were inoculated into tubes of SP-4 medium with fungizone and penicillin, and processed the same day as collection by diluting 1:10 in SP-4 medium containing fungizone and ciprofloxacin. The samples were passed through 0.45 μm-pore-size filters and serially diluted 1:1000 in SP-4 broth. All dilutions were incubated at 37oC in 10% CO2 for 1 month. Cultures exhibiting evidence of growth as determined by acidic pH change and microscopic detection of surface-adhering colonies were inoculated onto SP-4 agar plates and incubated at 37oC for 2 to 3 weeks. Colonies of M. genitalium were identified by hemadsorption of sheep erythrocytes, fluorescent antibodies, and PCR.

Cervical and vaginal swab specimens stored at –80oC were thawed, centrifuged, the pellets suspended in water, and incubated on ice for 20 min. The samples were centrifuged again, the pellets suspended in water, and boiled for 15 min. Samples were amplified using primers Mg1 and Mg2 that targeted the M. genitalium P140 adhesin gene. M. genitalium specific amplicons were identified by Southern blot analysis using P32-labeled probe MG-1.

Serum samples were tested for M. genitalium antibodies using an ELISA assay and an immunoblot. For the ELISA, lipid-associated membrane proteins (LAMP) from M. genitalium were extracted, diluted, and added to each well of microtiter plates, which were blocked with horse serum. The patients’ serum samples were added to duplicate wells and incubated for 2 h. Goat anti-human IgG conjugated to alkaline phosphatase and a colorimetric substrate were added. The cut-off for the assay (0.28) was determined by calculating the mean of the optical densities (OD) at 405 nm for the negative control sera plus 3 standard deviations. For the immunoblot, LAMP was separated by SDS polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Diluted serum samples were added to antigen-containing strips and incubated for 2 h. Goat anti-human IgG conjugated to alkaline phosphatase and a colorimetric substrate were added. Results were scored as negative, 1+, 2+, and 3+.

Main Outcome Measures
Using M. genitalium culture-positive status as the gold standard, the number of women who were positive by immunoblot, ELISA, or PCR at the time of culture and during the follow-up period was determined.

Main Results
Although over 3670 cervical and vaginal samples from 838 women were screened during the 17 to 39-month period of the study, the primary isolation and single-colony cloning of M. genitalium was accomplished from 31 women over a single 6-month interval and was not duplicated since that time. Among the 31 culture-positive women tested, M. genitalium was cultured from 26 cervical specimens and 27 vaginal specimens.

By the ELISA assay, 52 (96.3%) of the 54 control sera had ODs below the cutoff, while 16 (51.6%) of the 31 sera collected at time of culture from M. genitalium culture-positive women had ODs above the cutoff. Twenty-five (80.6%) of the culture-positive women seroconverted during the course of the study. Of 29 women with sufficient serum samples to evaluate, 13%, 32%, and 26% exhibited peak seroconversion prior to, at the time of, and after positive culture, respectively. All 31 women at the time of positive M. genitalium culture exhibited positive immunoblots. The two negative control sera with positive ELISA results were also positive by immunoblot.

Twelve (38.7%) of 31 women were M. genitalium PCR positive at time of culture. Twenty-five (80.6%) women were PCR positive for at least one sample during the course of the study. Among women positive by PCR, 75% were ELISA positive at the time of culture, and 84% were ELISA positive during the study period. Twelve (38.7%) women were PCR and ELISA negative at the time of culture. Two of the 6 women who remained PCR negative throughout the study also remained negative by the ELISA assay. The number of women with each immunoblot score, the ELISA OD median and range, and the number of PCR positive women with each score are shown in the table.

Results of ELISA assay for M. genitalium antibodies and PCR for M. genitalium DNA among 31 M. genitalium culture positive women by immunoblot score at time of culture.

Authors’ Conclusions
While the immunoblot assay was 100% sensitive for detection of M. genitalium, neither PCR nor ELISA provided the sensitivity needed to confidently confirm the presence of viable M. genitalium. The combined use of the two tests failed to achieve a sensitivity of 65% at the time of positive culture.

Source of funding: Grants from the National Institutes of Health and the Institute of Allergy and Infectious Disease