Literature review > Issue 8 > Review on Blaylock et al. 

M. genitalium is gaining increasing interest as a sexually transmitted infection causing urethritis in both men and women and cervicitis in women. A few studies have shown that M. genitalium may cause upper genital tract infection (PID) in women and indirect evidence that M. genitalium is associated with tubal factor infertility stems from a serological study. Because of the clinical importance of the infection, basic research in pathogenesis is needed.

The present study used confocal immunoanalysis (CIA) and real-time quantitative PCR to evaluate infectious patterns in vaginal swab specimens from a highly selected group of high-risk minority women attending an STD clinic in San Antonio, Texas.

Among 31 women with positive culture for M. genitalium [1], vaginal swab specimens from 18 women were selected based on PCR and enzyme-linked immunosorbent assay (ELISA) results. Probably these 18 were PCR positive and ELISA positive, but this is not described. Before being processed for PCR and CIA, the clinical specimens were washed three times by low-speed centrifugation and resuspension in PBS with the stated aim of removing non-adhering bacteria and fibres from the swab. Consequently, non-adhering M. genitalium were probably also washed away and the DNA load would therefore not represent that of the unprocessed vaginal secretion.

In a model study, Hep-2 cells were inoculated with different ratios of M. genitalium cells to human cells and incubated for 24 to 48 hours before they were used. DNA load from human cells was determined with a commercially available kit whereas an M. genitalium specific PCR detecting the gyrase gene was developed. However, the primer sequences as they are described in the paper could not generate a PCR product from M. genitalium. The forward primer contains a mismatch of 5 bases from the 5’ end (a C instead of a T). It would probably work despite this error. The reverse primer, however, did not match the M. genitalium genome sequence even when a significant number of mismatches were allowed. It appears that the sequence in the paper corresponds to complementary DNA strand, but written in the 3’ to 5’ direction. The probe sequence is correct.

In an attempt to document viability of the M. genitalium cells in the clinical specimens, a reverse transcriptase PCR assay detecting 16S rRNA was developed. As the specimens were selected based on being culture positive, this seems a bit redundant.

The main results were that there was a strong correlation between the M. genitalium DNA load and the CIA based scoring system (negative, positive, strongly positive). As the cells were washed before the quantitative PCR to remove non adhering M. genitalium cells, the result is not surprising. Interestingly, however, was the finding that in the same vaginal swab specimen, the vaginal cells exhibit a significant amount of variation in the number of M. genitalium cells per vaginal cell. This may reflect different subsets of vaginal cells or differences in age before desquamation. Also, it was found that in women heavily colonised, the number of vaginal cells containing intracellularly located bacteria was much higher. It would have been interesting to have observations on different cell types; i.e. were squamous epithelial cells more heavily infected than PMNLs or cervical cells? In the model system, most M. genitalium fluorescence was observed extracellularly regardless of the M. genitalium to Hep-2 cell ratio. This is probably reflecting that internalisation occurs relatively slowly and it would have been interesting to study the infected cells at a later time-point.

In conclusion the paper presents an interesting possibility for the study of M. genitalium in its natural environment, but in my opinion this opportunity has not been thoroughly exploited.

References

1. Baseman, J. B., M. Cagle, J. E. Korte, C. Herrera, W. G. Rasmussen, J. G. Baseman, R. Shain, and J. M. Piper. 2004. Diagnostic assessment of Mycoplasma genitalium in culture-positive women. Journal of Clinical Microbiology 42:203-211.

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