Literature review > Issue_1 > Review Kaydos et al. 

PCR-based methods have been shown to be more sensitive for the detection of T. vaginalis in women than traditional methods of wet mount or culture in many recent studies. These studies differ in many respects, including study populations, type of specimens used, specimen processing methods, primers used for the amplification reaction and methods for detection of amplicons. Even though most studies have shown that PCR assays using vaginal swabs have higher sensitivity than those using urine samples, in some clinical research settings where invasive sampling may be difficult or not possible, urine samples may be the only possible specimen for testing. For this reason, the authors sought to develop and validate a urine-based PCR method for detecting T. vaginalis in women against conventional wet mount and vaginal swab culture.

The authors selected a set of previously published primers for the PCR reaction and then described the results of PCR amplification using three amplicon detection methods with increasing sensitivity and/or specificity: gel electrophoresis, hybridization in an ELISA format with unlabeled probes, and hybridization with biotinylated probes and a digoxigenin-labeled primer. The performance of each PCR method was compared against the combined reference standard of wet mount and culture of vaginal swab specimens. This is a well designed and wonderfully described study with clearly stated objectives, sound and detailed methodology, adequate sample size, and thorough documentation of how the results were analysed and adjusted for a reference standard which is recognised to be imperfect. The conclusion that PCR combined with ELISA for detection of T. vaginalis in women's urine performed well compared to wet mount microscopy and culture from vaginal swabs is justified. The only shortcoming of this paper is that the data are not completely presented. The tables only contain computed sensitivity and specificity. The actual numbers of study subjects positive for each test are not given.

A useful piece of information arising from this study is that the urine volume and time since last void do affect the sensitivity of the PCR assay. This is not new or surprising but it is of interest in that only 186 of 780 (24%) women were able to comply with the investigators' request to collect 20 ml. of urine for testing. The same problem is often encountered when subjects are asked to collect first-void urine samples for PCR assays to detect other STD pathogens such as Chlamydia trachomatis and Neisseria gonorrhoeae. It is worth bearing that in mind when comparing the performance of urine-based versus vaginal swab based PCR methods. A diagnostic test can only be as good as the quality of specimen used for testing. Controlling the volume of urine collected for testing and maintaining a cold chain for transport remain a challenge for obtaining the best performance out of nucleic acid based amplification tests.

Another factor affecting the performance of PCR assays is the presence of inhibitors in clinical specimens. The authors mentioned in the discussion that they found a 9% inhibition rate for urine specimens in experiments using laboratory-grown organisms. The addition of an amplification control to monitor inhibition and then simple methods such as heating or dilution to remove the inhibitors would have possibly improved the performance of the PCR assays. It is not clear why the authors decided not to do that.

Overall, I enjoyed reading the paper very much and found the information to be useful and interesting. The methodology is developed for clinical research involving large scale population-based studies, and will be particularly useful in studies where urine specimens are being collected for the detection of other reproductive tract infections. I look forward to reading about their work on further improvements to the assay and validation of the test using specimens from men, which they alluded to in the paper.

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