Question
Can confocal immunoanalysis and real-time PCR be used to qualitatively and quantitatively assess patterns of M. genitalium infection?

Design
Confocal immunoanalysis (CIA) and real-time PCR were used to visually and analytically monitor frequency and infectious load of M. genitalium in vaginal specimens from M. genitalium culture-positive women attending an STD clinic.

Participants
Eighteen women, who were a subset of high-risk minority women enrolled in an ongoing longitudinal STD investigation at an STD clinic in San Antonio, Texas, were selected for testing based on M. genitalium culture-positive, PCR, and enzyme-linked immunosorbent results.

Description of Tests and Diagnostic Standard
Vaginal swab samples stored in 5 to 10 ml of buffer at 4^oC were centrifuged, and the pellets washed three times prior to suspension in 2 ml of buffer.  One ml was used for DNA isolation and one ml was used for CIA.  Quantification of M. genitalium genome numbers was achieved by real-time PCR using TaqMan primers and probe that amplified an 86 bp fragment of the gyrA gene.  The number of human cells was quantified by TaqMan amplification of the RNase P gene.  Standard curves were established by amplification of serially diluted M. genitalium strain G37 DNA from 10⁷ to 1 copy per reaction and human DNA diluted from 10⁸ to 1 copy per reaction.  Multiplex real-time PCR permitted measurement of the ratio of M. genitalium to human genomic DNA in order to quantify the dynamics of variations of mycoplasma-host cell interactions.  

Samples for CIA were permeabilized by Triton X-100 and fixed with neutral buffered formalin.  Polyclonal rabbit anti-M. genitalium strain G37 antiserum was added.  Secondary immunolabeling was performed with FITC-labeled goat anti-rabbit antibody.  Propidium iodide was added to stain nucleic acids.  Slides were viewed with an Olympus IX70 microscope and an Olympus FV500 confocal system.  Lasers were employed to visualize FITC and propidium iodide labeling patterns.  All PCR and CIA reactions were performed in triplicate.  For CIA, 20 fields of 8 to 10 cells (150 to 200 vaginal cells) per sample were examined to determine M. genitalium qualitative infectious patterns.  A representative group of 20 to 25 cells was selected for scoring of discrete immunofluorescence bodies.  The levels of infection were scored as negative (less than 3% of vaginal cells exhibited any fluorescence and no internalized fluorescent bodies visualized in a complete Ζ-series of individual cells), positive (approximately 3 to 30 % of vaginal cells exhibited fluorescence with 5 to 15 fluorescent bodies detected on surfaces and within individual cells), and strongly positive (30 to 100% of vaginal cells exhibited positive immunofluorescence with 16 or more fluorescent bodies detected on surfaces and within individual cells).  

Main Outcome Measures
The ratio of M. genitalium to human cells by PCR was compared to the level of M. genitalium parasitism (negative, positive, strongly positive) among individual vaginal cells by CIA.  

Main Results
The median ratio of M. genitalium to vaginal cells as determined by PCR was 0.14 for the CIA negative group, while the ratios were 0.65 and 3.21 for the CIA positive and strongly positive groups, respectively.  The test for trend in median M. genitalium/vaginal cell values across CIA categories was statistically significant (P<0.001).  The extent of M. genitalium parasitism varied from strongly positive to negative in vaginal cells within the same sample field.  Heavily parasitized strong-positive cells revealed considerably greater numbers of intracellular than surface-associated mycoplasmas.  Immunoelectron microscopy confirmed the intracellular presence and cell integrity of M. genitalium in vaginal samples.  

Authors' Conclusions
M. genitalium could be spatially localized on vaginal cell surfaces and intracellularly evaluated by CIA.  The correlation between real-time PCR values of mycoplasma/human genome ratios and CIA determinations was very strong and a statistically significant trend in this ratio across CIA categories was observed.  Either CIA or real-time PCR might be useful in identifying potentially high transmitters, assessing response to antimicrobial therapy, and evaluating levels of mycoplasma persistence, chronicity of infection, and immune competence in the host.  

Source of funding: Grants from the National Institute of Allergy and Infectious Diseases 

For correspondence:  Joel B. Baseman, Department of Microbiology and Immunology, The University of Texas Health Science Center, Mail Code 7758, 7703 Floyd Curl Dr., San Antonio, TX 78229-3900.  E-mail address: baseman@uthscsa.edu.