Determination of infectious load of Mycoplasma genitalium in clinical samples of human vaginal cells

Abstract

Mycoplasma genitalium is a leading cause of chlamydia-negative, nongonoccocal urethritis and has been directly implicated in numerous other genitourinary as well as extragenitourinary tract pathologies. Detection of M. genitalium has relied almost entirely on PCR amplification of clinical specimens and evidence of seroconversion since these mycoplasmas are highly fastidious and culture isolation by microbiological techniques is very rare. We have established a combinatorial strategy using confocal immunoanalysis (CIA) and real-time PCR to qualitatively and quantitatively assess patterns of M. genitalium infection in women attending a sexually transmitted disease-related health clinic in San Antonio, Tex. CIA allows spatial examination of mycoplasmas on surfaces and inside human target cells, plus the ability to evaluate cell-to-cell patterns and variances within samples. Real-time PCR permits determination of genome copy numbers of mycoplasmas and human cells by multiplex amplification using mycoplasma gyrA and human RNase P gene sequences, which indicates overall levels of mycoplasma infection and degree of parasitism. These assays are strongly correlated and, in combination, permit detection and elucidation of heretofore-unrecognized patterns of M. genitalium infections in clinical and experimental samples.

FIG. 1.

Sequential visualization of clinically derived vaginal cells using CIA. Both surface-associated and internalized immunolabeled M. genitalium cells are readily observed using 1-μm-diameter z-series optical sections. (A to F) Images are representative of a cell with a strong positive score. Mycoplasmas can be readily visualized as intense, discrete white fluorescing bodies, while the cell nucleus can be visualized using propidium iodide as detected (B to E).

FIG. 2.

Representative clinically derived vaginal cells based upon immunolabeled patterns of M. genitalium as described in Fig. 1 legend. Mycoplasmas appear as distinct fluorescent bodies as in Fig. 1, and the vaginal cell nucleus is clearly observed in each panel. CIA scores: negative (A), positive (B), and strongly positive (C).

FIG. 3.

Electron micrograph of epoxy-embedded immunolabeled clinically derived vaginal cell sample. (A) Intracellular M. genitalium cells labeled with 10-nm-diameter immunogold beads appear as two nearly complete bisected bodies and two smaller ones, each surrounded by a unit membrane and associated with gold beads. (B′) Enlarged inset of panel B, revealing intact mycoplasma unit membrane. Bar = 500 nm.

FIG. 4.

Reverse transcription-PCR of M. genitalium G37 control and individual clinical vaginal specimens. In lanes G37, A, B, and C, positive lanes (+) indicate M. genitalium 16S rRNA 517-bp fragment amplification with addition of reverse transcriptase; negative lanes (-) are without reverse transcriptase. Vaginal cells from patients A, B, and C scored positive using immunofluorescent confocal microscopy while negative sample D showed no amplification.