Literature review > Issue 7 > Review on Dutro et al. 

Although Mycoplasma genitalium has been associated with genitourinary tract diseases such as urethritis in men [1] and cervicitis in women [2], it has not yet been elevated to the pathogen status of Chlamydia trachomatis and Neisseria gonorrhoeae for widespread testing of symptomatic or asymptomatic patients. Totten and her associates provide an excellent review of the literature concerning the association of M. genitalium and reproductive tract disease in the introduction section of this paper. However, they continue to stress the need for additional clinical studies to elucidate the role of M. genitalium in pelvic inflammatory disease, ectopic pregnancy, and other potential disease sequelae. In addition, large epidemiology studies are needed to assess appropriate treatment regimens for M. genitalium infection.

Culture of M. genitalium is extremely difficult and time-consuming. A number of different Southern blot and gel-based PCR tests have been described in the literature, but they are also lengthy (4 days) and not useful for large-scale screening of patient specimens. A 96-well microtiter PCR method is described here in great detail, along with the addition of an internal control to detect sample inhibition to the PCR. The method is faster (2 days) and more appropriate for screening large groups of patient specimens (up to 82 samples and 6 controls) compared to a previously used Southern blot-based PCR test. It employs a different themocycler (9600), shorter cycle times, and an entirely different colorimetric detection scheme.

The design of the study was complicated, with a number of variables to consider. At times this makes the paper difficult to read and follow. The new PCR assay was compared both analytically and clinically to a previously published Southern blot-based PCR method. Both methods were tested with and without an internal control added. The clinical samples (cervical swabs and urine) were either tested fresh or frozen (up to 15 years!). The urine samples were processed by 3 different purification methods, and the amount of processed specimen going into the PCR amplification varied. In addition, the methods for discrepancy resolution varied, including: repeat tests, dilution of the specimen 1:5 followed by repeat testing, re-purification of the specimen followed by repeat testing, and freeze-thaw of the specimen followed by repeat testing.

In spite of all these variables, the new PCR test was remarkable in several ways. First, the addition of the internal control did not affect the excellent analytical limit of detection for M. genitalium (approximately 17 genome copies of DNA). Second, the results for the clinical specimens were essentially equivalent for the two PCR methods studied: 97% concordance for the urine samples and 100% for the cervical samples following discrepancy analysis. Third, two of the three urine processing methods had 95% concordance. Fourth, the detection method gives a numeric cutoff value for clear interpretation of results, rather than subjective evaluation of bands on a gel.

The addition of analytical specificity data (or a reference to it) would have added to the completeness of this assay evaluation. How does the assay perform with large quantities of Mycoplasma hominis or other closely related bacteria? And, how does the test perform with a wide variety of M. genitalium strains?

Prospective trials, using fresh specimens and a final, optimized approach for specimen processing, would reduce the number of variables. A more consistent approach to discrepancy testing would also be useful.

Overall, this new PCR method has been well-characterized, and should be a very useful tool to further elucidate the epidemiology and pathogenicity and treatment aspects of M. genitalium infection

References:

1. Totten PA, Schwartz MA, Sjostrom KE, Kenny GE, Handsfield HH, Weiss JB, Whittington WL. Association of Mycoplasma genitalium with nongonococcal urethritis in heterosexual men.
J Infect Dis. 2001 Jan 15;183(2):269-276.

2. Manhart LE, Critchlow CW, Holmes KK, Dutro SM, Eschenbach DA, Stevens CE, Totten PA. Mucopurulent cervicitis and Mycoplasma genitalium.
J Infect Dis. 2003 Feb 15;187(4):650-7.

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